Method for generating oligonucleotides, in particular for the detection of amplified restriction fragments obtained using AFLP®

ABSTRACT

The invention relates to a method for generating, and optionally detecting, an oligonucleotide, comprising at least the steps of: a) providing a first dsDNA; b) ligating the first dsDNA to a second dsDNA, in which said second dsDNA sequence comprises within its sequence at least one recognition site for an IIS restriction endonuclease; so as to provide a ligated dsDNA; d) restricting the ligated dsDNA with the at least one restriction endonuclease of the IIS type so as to obtain at least a first and a second IIS-restricted dsDNA; and optionally comprising the further step of: e) detecting the first and/or second IIS-restricted dsDNA obtained in the restriction step d). Preferably, said method comprises the further step of: c) amplifying the ligated dsDNA obtained in the ligating step b) prior to the restriction step d). The detection step e) preferably comprises the steps of: 1) generating at least one ssDNA from either the first or the second IIS-restricted dsDNA obtained in the restriction step d); 2) detecting the at least one ssDNA generated from either the first or the second IIS-restricted dsDNA; and is more preferably based upon the molecular mass of, and/or the nucleotide sequence of, the fragment(s) obtained in the restriction step d), such as a mass spectroscopy technique, in particular of MALDI-TOF; and/or a chromatography technique, such as high pressure liquid chromatography (HPLC); or a suitable combination thereof, such as Gas Chromatography-Mass Spectroscopy (GC-MS).

The present invention relates to a method for generating, and optionallydetecting, oligonucleotides.

In particular, the present invention relates to a method for generating,from any desired starting DNA, DNA fragment or mixture of DNAs or DNAfragments, oligonucleotides of a known and predetermined length whichare specific for said starting DNA(s) or DNA fragment(s) and which cansuitably be detected using mass spectroscopy or a similar detectiontechnique.

According a preferred embodiment, the method of the invention can beused to detect amplified restriction fragments obtained using AFLP®and/or to analyse a mixture of amplified restriction fragments obtainedusing AFLP®

Selective restriction fragment amplification or AFLP® is known, forinstance from the European patent application 0 534 858 by applicant,incorporated herein by reference. In general, AFLP® comprises the stepsof:

-   (A) digesting a nucleic acid, in particular a DNA, with one or more    specific restriction endonucleases, to fragment said DNA into a    corresponding series of restriction fragments;-   (B) ligating the restriction fragments thus obtained with at least    one doubled-stranded synthetic oligonucleotide adapter, one end of    which is compatible with one or both of the ends of the restriction    fragments, to thereby produce tagged restriction fragments of the    starting DNA;-   (C) contacting said tagged restriction fragments under hybridising    conditions with at least one oligonucleotide primer;-   (D) amplifying said tagged restriction fragments hybridised with    said primers by PCR or a similar technique so as to cause further    elongation of the hybridised primers along the restriction fragments    of the starting DNA to which said primers hybridised; and-   (E) identifying or recovering the amplified or elongated DNA    fragment thus obtained.

According to the prior art, the amplified DNA-fragments thus obtainedcan then be analysed and/or visualised, for instance by means ofgel-electrophoresis. This provides a genetic fingerprint showingspecific bands corresponding to the restriction fragments which havebeen linked to the adapter, have been recognised by the primer, and thushave been amplified during the amplification step. The fingerprint thusobtained provides information on the specific restriction site patternof the starting DNA, and thus on the genetic make-up of the organismfrom which said DNA bas been derived.

AFLP® can therefore be used to identify said DNA; to analyse it for thepresence of specific restriction site patterns, restriction fragmentlength polymorphism's (RFLP's) and/or specific genetic markers(so-called “AFLP-markers”), which may be indicative of the presence ofcertain genes or genetic traits; or for similar purposes, for instanceby comparing the results obtained using DNA-samples of known origin orrestriction pattern, or data thereon.

The primers used in AFLP® are such that they recognise the adapter andcan serve as a starting point for the polymerase chain reaction. To thisend, the primers must have a nucleotide sequence that can hybridise with(at least part of) the nucleotide sequence of the adapters(s) ligated tothe end(s) of the restriction fragment to be amplified. The primers canalso contain one or more further bases (called “selective bases”) at the3′-end of their sequence, for hybridisation with any complementary baseor bases at the corresponding positions in the adapter ligatedrestriction fragment, i.e. adjacent to the adapter(s) and therestriction site(s). As, of all the adapter ligated restrictionfragments present in the mixture, only those fragments that containbases complementary to the selective bases will subsequently beamplified efficiently, the use of these “selective” primers will reducethe total amount of bands in the final fingerprint, thus making thefingerprint more clear and more specific. Also, the use of differentselective primers will generally provide differing fingerprints, whichcan also be used as a tool for the purposes of identification oranalysis.

As AFLP® provides amplification of both strands of a double strandedstarting DNA, AFLP® advantageously allows for exponential amplificationof the fragment, i.e. theoretically according to the series 2, 4, 8, 16,etc. Also, AFLP® requires no prior knowledge of the DNA sequence to beanalysed, nor prior identification of suitable probes and/or theconstruction of a gene library from the starting DNA.

For a further description of AFLP®, its advantages, its embodiments, aswell as the techniques, enzymes, adapters, primers and further compoundsand tools used therein, reference is made to EP-0 534 858, incorporatedherein by reference. Also, in the description hereinbelow, thedefinitions given in paragraph 5.1 of EP-0 534 858 will be used, unlessindicated otherwise.

Although AFLP® is generally less time-consuming than otherhybridisation-based techniques such as PCR-based detection, it stillsuffers from the disadvantage that the amplified fragments have to beseparated (i.e. by (gel-) electrophoresis) and visualised (i.e. bygeneration of a fingerprint). These very elaborate and time-consumingprocedures, which require special apparatus, such as electrophoresis andautoradiography equipment. Thereafter, the fingerprints have to beanalysed—nowadays generally performed by “reading” the fingerprint intoa computer—to identify the polymorphic bands. Generally, this alsorequires using a known reference sample run at the same time in aparallel lane of the gel.

Because of these factors, AFLP® can only be carried out in sufficientlyequipped laboratories. Even so, it may take several days until resultsare obtained, even when routine tests following known protocols arecarried out, such as on species or individuals of which the genomeand/or relevant AFLP®-markers are generally known.

A first aim of the invention is to simplify these procedures, i.e. toprovide a technique analysing nucleic acid sequences, which no longerrequires the use of (gel-) electrophoresis and/or autoradiography.

The use of mass-spectroscopy techniques such as matrix assisted laserdesorption/ionisation time-of-fight (MALDI-TOF) fordetecting/identifying single strand DNA fragments is known, for instancefrom WO 97/47766; WO 99/54571; WO 99/02728; WO 97/33000, as well asGriffin et al., Proc. Natl. Acad. Sci. USA., Vol. 96, pp. 6301–6306(1999); Ross et al., Nature Biotechnology, Vol. 16 (1998), p. 1347–1351;and Berkenkamp et al., Science, Vol. 281 (1998), p. 260–262.

However, MALDI-TOF generally cannot be used to identify/detect therestriction fragments obtained using AFLP®, i.e. the fragments obtainedin step (d) above. This is because MALI-TOF is limited to detection ofsingle strand (hereinbelow further indicated as “ss”) oligonucleotideswith a length of at most 100, and preferably at most 30 bases, whereasthe AFLP®-generated amplified fragments are mostly double stranded(hereinbelow indicated as “ds”) DNA fragments which typically have alength in the range of 50–1200 base pairs.

Mass spectroscopy techniques have been used for theidentification/detection of oligonucleotide sequences generated usingPCR-amplification. In these methods, a starting DNA is amplified by aPCR using primers that are specifically designed to hybridise with thestarting DNA at a site/sequence that is located in close proximity to a(single nucleotide) polymorphism (SNP) and that contain a recognitionsite for a restriction endonuclease of the IIS-type. Following the PCR,the amplified DNA is restricted with the corresponding IIS restrictionendonuclease to generate small ds oligonucleotide fragments, which areconverted to a corresponding ssDNA and detected using mass spectroscopy.Such a technique—using ESI-MS—is for instance described by Laken et al.,Nature Biotechnology, vol. 16 (1998), p. 1352–1356.

However, these techniques suffer from the general disadvantage ofPCR-based techniques, in that at least some prior knowledge of thesequence to be analysed is required, i.e. sufficient to provide a primerthat can hybridise with the starting DNA.

By comparison, AFLP® requires no such prior knowledge of the sequence tobe analysed. Thus, another objective of the invention is therefore toprovide a method which combines the advantages of AFLP® with the easeand high throughput of detection that can be achieved using MALDI-TOF ora similar detection technique. This objective is achieved by themethod(s) described hereinbelow.

In a first aspect, the invention relates to a method for generating, andoptionally detecting, an oligonucleotide, comprising at least the stepsof:

-   a) providing a first dsDNA;-   b) ligating the first dsDNA to a second ds DNA, in which said second    dsDNA sequence comprises within its sequence at least one (sequence    corresponding to a) recognition site for an IIS restriction    endonuclease; so as to provide a ligated dsDNA;-   d) restricting the ligated dsDNA with the at least one restriction    endonuclease of the IIS type so as to obtain at least a first and a    second IIS-restricted dsDNA;    and optionally comprises the further step of:-   e) detecting the first and/or the second IIS-restricted dsDNA    obtained in the restriction step d).

Optionally, this method comprises the further step of:

-   c) amplifying the ligated ds DNA obtained in the ligating step b)    prior to the restriction step d);

Further aspects, advantages and embodiments of the invention will becomeclear from the description given below.

Preferably, the detection step e) comprises the following steps:

-   e1) generating at least one ssDNA from either the first or the    second IIS-restricted dsDNA obtained in the restriction step d);-   e2) detecting the at least one ssDNA generated from either the first    or the second IIS-restricted dsDNA.

The detection step e) preferably involves a detection method based uponthe sequence and/or the molecular mass of the dsDNA obtained in therestriction step d). According to the preferred embodiment involvingsteps e1) and e2), the detection step e) involves a detection methodbased upon the molecular mass of the ssDNA generated in step e1).

In particular, mass spectroscopy techniques, and more in particularMALDI-TOF; and/or chromatography techniques, more in particular highpressure liquid chromatography (HPLC) may be used; or a suitablecombination thereof.

In the Figures:

FIGS. 1 to 3 are schematic drawings illustrating the method of theinvention for generating detectable oligonucleotides.

FIGS. 4 and 5 are DNA fingerprints obtained in Example 1 and Example 2,respectively.

FIG. 6 schematically shows an alternative method of the invention forgenerating detectable oligonucleotides using an IIS-type restrictionendonuclease that is capable of restricting dsDNA at two sites differentfrom the recognition site.

FIG. 7 shows AFLP reactions prepared with a MseI adapter- and AFLPprimers containing a GsuI site.

FIG. 8 shows AFLP reactions prepared with a MseI adapter- and AFLPprimers containing a BsgI site.

FIG. 9 shows AFLP reactions prepared from templates prepared with theBcgI restriction enzyme.

FIG. 10 is a mass spectrum from a Maldi TOF — Mass spectrophotometricanalysis performed on oligonucleotide tags.

Usually, the first dsDNA will be a naturally occurring DNA or DNAfragment (including genomic DNA and/or a fragment thereof); a cDNA or acDNA fragment; and/or an amplified DNA or DNA fragment (althoughpreferably—as in step c) above—an amplification step forms part of themethod of the invention). As such, the first dsDNA or dsDNA fragment maybe part of a mixture of such dsDNAs or dsDNA fragments.

Preferably, as mentioned below, the first dsDNA is a restrictionfragment. As such may be part of a mixture of restriction fragmentsobtained by restricting a starting DNA, and in particular a genomic DNAor cDNA, with one or more restriction enzymes.

Also, as further described below, when the starting dsDNA or dsDNAfragment is part of a mixture or sample containing several such dsDNAsor dsDNA fragments, it is possible, using the method of the invention,to generate—e.g. simultaneously and/or in a singlereaction—oligonucleotides from (i.e. specific for) only one of thedsDNAs or dsDNA fragments present in the mixture or sample; from severalof the dsDNAs or dsDNA fragments present in the mixture or sample; froma specific subset of the dsDNAs or dsDNA fragments present in themixture or sample; or from essentially all of the dsDNAs or dsDNAfragments present in the mixture. In such a case, according to theinvention, it may be possible to separately/independently detect (eachof) the oligonucleotide(s) specific for each of the dsDNAs or dsDNAfragments present in the original mixture or sample.

Thus, the method of the invention may be used to analyse such a sampleor mixture, for instance for determining the presence or absence of oneor more specific dsDNAs and/or for distinguishing between one or morespecific dsDNAs, in which the one or more dsDNAs may for instance be oneor more restriction fragments corresponding to one or more geneticmarkers of interest.

The second dsDNA will comprise within its sequence at least arecognition site for an IIS restriction endonuclease. Herein, by an IIS(restriction) endonuclease is meant an endonuclease that restricts adsDNA at a site/position on the dsDNA different from its recognitionsite, and in particular endonuclease that restricts a dsDNA at a certain“distance” from the recognition site, usually from 5 to 30 base pairs,and preferably more than 15 base pairs, from the recognition site, inwhich said “distance” is defined as the number of base pairs situatedbetween “last” base(pair) of the recognition site and “first” base pairof the restriction site.

Some non-limiting examples of type IIS endonucleases suitable for use inthe invention and their recognition site(s) are mentioned in Table 1:

Endonuclease Recognition site MmeI 5′ TCCRAC(N)₂₀ 3′ (SEQ ID NO.41)3′ AGGYTG(N)₁₈ 5′ (SEQ ID NO.42) Eco57I 5′ CTGAAG(N)₁₆ 3′ (SEQ ID NO.43)3′ GACTTC(N)₁₄ 5′ (SEQ ID NO.44) BsgI 5′ GTGCAG(N)₁₆ 3′ (SEQ ID NO.45)3′ CACGTC(N)₁₄ 5′ (SEQ ID NO.46) GsuI 5′ CTGGAG(N)₁₆ 3′ (SEQ ID NO.47)3′ GACCTC(N)₁₄ 5′ (SEQ ID NO.48) TaqII 5′ CACCCA(N)₁₁ 3′ (SEQ ID NO.49)3′ GTGGGT(N)₉ 5′ (SEQ ID NO.50) Tth111II 5′ CAARCA(N)₁₁ 3′ (SEQ IDNO.51) 3′ GTTYGT(N)₉ 5′ (SEQ ID NO.52) BsmFI 5′ GGGAC(N)₁₀ 3′ (SEQ IDNO.53) 3′ CCCTG(N)₁₄ 5′ (SEQ ID NO.54) FokI 5′ GGATG(N)₉ 3′ (SEQ IDNO.55) 3′ CCTAC(N)₁₃ 5′ (SEQ ID NO.56) BbvI 5′ GCAGC(N)₈ 3′ (SEQ IDNO.57) 3′ CGTCG(N)₁₂ 5′ (SEQ ID NO.58)

Other suitable IIS restriction enzymes will be clear to the skilledperson and are for instance mentioned in U.S. Pat. No. 5,658,736 and WO98/48047 and include e.g. AceIII, AlwI, AlwXI, Alw26I, BbvI, BbvII,BbsI, BccI, Bce83I, BcefI, BcgI, BinI, BsaI, BsgI, BsmAI, BsmF1, BspMI,EarI, EciI, Eco31I, Eco57I, Esp3I, FauI, FokI, GsuI, HgaI, HinGUII,HphI, Ksp632I, MboII, MmeI, MnlI, NgoVIII, PleI, RleAI, SapI, SfaNI,TaqII and Tth111II.

The second dsDNA will usually have a known and predetermined“size”/“length” (by which is meant the total number of basepairs/nucleotides thereof). Also, the position of the IIS recognitionsite in the second dsDNA will usually be known and pre-determined andwill be such that, in the ligated DNA obtained in step b), therestriction site of the IIS endonuclease lies in the part of the ligatedDNA that has been derived from/corresponds to the dsDNA. If so, therestriction step c), two IIS-restricted dsDNA fragments will beobtained, i.e. one containing the second dsDNA (with the IIS recognitionsite) and a part of the first dsDNA (i.e. that in the ligated dsDNA wasdirectly adjacent to the second dsDNA); and one containing the remainderof the first dsDNA. Of these two IIS restricted fragments, for reasonsthat will be further explained hereinbelow, in step e) preferably thefragment comprising (a sequence corresponding to) the second dsDNA whichincludes the IIS recognition site is detected. More preferably, in stepe1), the at least one ssDNA is preferably generated from the fragmentcomprising (a sequence corresponding to) the second dsDNA, which issubsequently detected.

Usually, the second dsDNA will have a size/length of up to 20–50 basepairs, and preferably between 10 base pairs and 40 base pairs. Thesecond dsDNA will usually also have at least one “end” that allows it tobe ligated to the first dsDNA—e.g. using conventional DNA ligationprotocols—which will usually depend upon the end(s) present on the firstdsDNA. Preferably, the IIS recognition site will be at a “distance” fromthe end of the second dsDNA that is to be ligated to the first dsDNA (bywhich is meant the number of base pairs/nucleotides between the IISrecognition site and the end of the second dsDNA) of up to 6–10 basepairs, and preferably between 0 and 6 base pairs, depending upon the IISendonuclease to be used.

Usually, the second dsDNA will be a synthetic sequence, although theinvention is not limited thereto. According to one preferred embodiment,the second dsDNA essentially corresponds to, and/or is used in a manneressentially analogous to, a conventional dsAFLP® adapter sequence.

Also, when the method of the invention comprises an amplification stepc), this is preferably carried out using at least one primer that canhybridise with that part of the second dsDNA that allows extension ofthe primer along the ligated dsDNA. More preferably, this primercontains a IIS restriction site as close a possible to its 3′ prime end,so as to yield—after restriction with the appropriate IIS restrictionendonuclease—an IIS restricted dsDNA with as many as possiblenucleotides derived from the first dsDNA.

Also, the primer is preferably such that it can also hybridise with 1–10bases, preferably 1–4 bases, of the first dsDNA that, in the ligateddsDNA obtained in step b), are immediately adjacent to the sequencederived from the second dsDNA, so as to allow efficient amplification ofonly as subset of ligated dsDNA fragments when present in a mixturecontaining multiple such ligated dsDNA fragments).

However, the invention is not limited thereto, but generally comprisesthe use of any primer that allows for extension along the ligated dsDNA.For instance, the invention also encompasses the use of a primer thatcan hybridise with the sequence of the second dsDNA—i.e. as present inthe ligated dsDNA used as the template for the extension reaction—suchthat, upon hybridisation, the 3′-end of the primer is still some basesremoved from the restriction site (e.g. the sequence corresponding tothe first dsDNA).

As further described below, such a primer will essentially correspond toan AFLP® primer and, in its preferred embodiment, to a selectiveAFLP®-primer. As with selective AFLP® primers, which “selectivenucleotides”are used—i.e. the specific (sequence of) nucleotides presentin the primer that are to hybridise with the bases/nucleotides derivedfrom the first dsDNA—is not essential, as long as (at least a degree of)selective amplification is obtained, i.e. amplification of only a subsetof all ligated dsDNA(s) present in the sample or mixture. However, as inconventional AFLP, the selective nucleotides are preferablypredetermined.

The invention will now be explained in more detail with reference to thenon-limiting FIG. 1, which schematically shows the method of theinvention.

In step b), a first dsDNA (1) is ligated to a second dsDNA (2)containing an IIS recognition site (3). The ligated dsDNA (4) thusobtained is restricted in step c) with the appropriate IIS restrictionendonuclease, which cuts the ligated dsDNA (4) at a restriction site (5)which lies in the part of the ligated dsDNA (4) that has been derivedfrom the first dsDNA (1). The position of the restriction site (5)relative to recognition site (3)—i.e. the “distance” betweensites—essentially will be determined by the IIS restriction endonucleaseused.

Upon restriction, two IIS restricted dsDNA fragments will be obtained,indicated as (6) and (7) in FIG. 1. One of these fragments—indicated as(6) in FIG. 1—will comprise the original second dsDNA with the IISrestriction site and part of the first dsDNA, whereas the otherfragment—indicated as (7) in FIG. 1—will comprise the remainder of theoriginal first dsDNA.

It should be clear that the total “length”/“size” of fragment (6) willbe determined by the length of the second dsDNA (2), the position of theIIS-recognition site (3) therein, and by the “distance” from the IISrecognition site (3) that the IIS restriction endonuclease restricts theligated dsDNA (4), i.e. the position of site (5). Therefore, by using asecond dsDNA (2) of known and pre-determined length and with a known andpre-determined position of the IIS recognition site (3), an IISrestricted dsDNA fragment (6) can be obtained of known and predeterminedlength, the mass of which—respective of similar fragments (6) generatedwith the same second dsDNA and using the same IIS restrictionendonuclease—will essentially be determined by the nucleotides/basesfrom the first dsDNA (1) present in the IIS restricted fragment (6).Thus, the mass and/or the nucleotide sequence of the IIS restrictedfragments (6) will be indicative of/characteristic for the first dsDNA(1).

Preferably, of the nucleotides/base pairs present in the IIS restricteddsDNA fragment(s) (6) thus obtained, at least 8 nucleotides/base pairs,more preferably at least 10 nucleotides base pairs, and most preferablyup to the maximum number of base pairs/nucleotides that can be achievedwith the restriction endonuclease used, are derived from the firstdsDNA. This increases the difference(s) in mass between the differentIIS restricted dsDNA fragment(s) (6) that have been generated from thestarting DNA using the method of the invention, which improvesresolution/sensitivity.

The IIS-restricted fragment (6) may then be detected, for instance by adetection method based upon (differences in) molecular weight/massand/or nucleotide sequence. According to the preferred embodiment of theinvention, this is carried out by generating—i.e. in step d)—at leastone ssDNA fragment (8) from the IIS—restricted fragment (6), anddetecting said ssDNA fragments (8) by mass spectroscopy or a similartechnique, most preferably by MALDI-TOF. For this purpose, thelength/size of the ssDNA fragment (8) generated from dsDNA fragment (6)should be such that the fragment is suitable for detection by MALDI-TOF,i.e. be within the general range of up to a maximum of 10–100nucleotides, and preferably between 1 nucleotide and 40 nucleotides.

Conveniently, the size of the ssDNA fragments (8) obtained can be (andis) determined by appropriate selection of (the size/length of) thesecond dsDNA (2), i.e. such that the IIS restricted dsDNA fragment (8)has a size that—after separation of the strands—directly yields ssDNAfragments (8) of suitable length/size. However, it is not excluded that,as part of the steps by which the ssDNA fragment (8) is generated fromthe dsDNA fragment (6), the length/size of the ssDNA fragment (8)obtained is reduced further, i.e. compared to the length/size of thedsDNA fragment (6), for instance to afford an ssDNA fragment (8) ofsuitable length for detection by MALDI-TOF from a larger dsDNA fragment(6); provided that the ssDNA fragment (8) obtained is stillspecific/characteristic for the first dsDNA (1).

The fragment(s), and in particular the ssDNA fragment(s) (8), may thenbe detected using a mass spectroscopy technique, and in particular amass spectroscopy technique suitable for the detection of nucleic acids.

Generally, such a technique will involve an ionisation step and adetection step. The ionisation step may for instance be carried outusing electronspray ionisation (ESI) or matrix assisted laser desorptionionisation (MALDI). The detection step may for instance be carried outusing ion trap (IT), time-of-flight (TOF), or quadrupole, or Fouriertransform ion cyclotron resonance (FTICR). Any suitable combination ofsuch an ionisation and such a detection technique may be used, of whichESI-quadrupole, ESI-FTICR and MALDI-TOF are the most commonly used. Inthe invention, the use of MALDI-TOF is particularly preferred.

The mass spectroscopy may be carried out according to known protocols,and the spectrum obtained may be analysed in a manner known per se.

For a further description of the above and other suitable massspectroscopy techniques and their use in analysing nucleic acidsequences, reference is inter alia made to WO 97/47766; WO 99/02728; WO97/33000.

One advantage of the method of the invention is that the use of massspectroscopy labels—although not excluded from the scope of theinvention—is not required.

However, with respect to the detection of the IIS restricted fragmentsobtained in step (d) above—i.e. of fragment (6) and/or (7) in FIG. 1—itshould be noted that the above represents a non-limiting preferredembodiment, and that other ways of detecting/analysing the IISrestricted fragments are possible.

For instance, instead of generating a ssDNA (8) from the IIS-restricteddsDNA fragment (6), it may also be possible to detect dsDNA fragment (6)directly. Also, it may be possible to detect the IIS-restricted dsDNAfragment (7) and/or a ssDNA generated therefrom (not shown in FIG. 1).

Also, although the use of mass spectroscopy (MS) based detectiontechniques, and in particular of MALDI-TOF is preferred, it may bepossible to use other detection techniques such as chromatographytechniques such as HPLC or gas chromatography (GC) techniques; or asuitable combination of chromatography and mass spectroscopy techniques,such as GC MS.

The preferred techniques via which the ssDNA fragment(s) (8) can beobtained from the IIS restricted dsDNA fragment (6) will be furtherdiscussed hereinbelow.

The invention will now be further illustrated with reference to itspreferred embodiment and application, i.e. the detection of restrictionfragments, more specifically the combined amplification and detection ofrestriction fragments using AFLP® methodology. In this aspect, the firstdsDNA (1) will be a restriction fragment, usually present in a mixtureof restriction fragments, e.g. generated as described hereinbelow. Thesecond dsDNA (2) containing the IIS restriction site will correspond toan AFLP® adapter, i.e. be designed such that it has an IIS recognitionsite in its sequence.

Also, in this aspect of the invention, the method of steps a) to e)above will usually also comprise an amplification step c). Conveniently,for the purposes of said amplification, use will be made of the AFLP®adapter containing the IIS recognition site, and of AFLP®-primerscorresponding thereto. Thus, in the invention, the adapter is used bothto provide a suitable IIS recognition site as well as to provide foramplification of the restriction fragments.

This has the advantage that the method of the invention can convenientlybe carried out as a conventional AFLP®-reaction/amplification, i.e.essentially according to steps (A) to (D) of EP 0 534 958 mentionedabove or according to another AFLP® protocol known per se, in whichsteps (A) to (D) will essentially correspond to steps a) to c) of themethod of the invention. For this, the only adaptation required comparedto conventional AFLP® methodology will be the use of an adaptercontaining an IIS restriction site, which can further be used in amanner per se for AFLP® adapters.

Thereafter, the detection of the amplified restriction fragments—step(E) of the method of EP 0 534 858—is carried out in accordance with themethod of the invention described above, i.e. according to steps d) ande) above. In essence, this means that the amplified mixture ofrestriction fragments is treated with the suitable IIS restrictionendonuclease, upon which—preferably—the amplified ds restrictionfragments are converted into ss fragments, which can then be detected,e.g. using MALDI-TOF. Thus, steps d) and e) of the present inventionwill replace gel electrophoresis and autoradiography in conventionalAFLP®.

Furthermore, as the AFLP® amplification itself can be carried out in amanner known per se, the present invention can incorporate and benefitfrom all advantages of AFLP®, as well as all the embodiments of AFLP®.For instance, as in conventional AFLP®, the amplification step c)—i.e.step (D) of EP 0 534 858—can be carried out using selective primers, inorder to selectively amplify only a subset of all the restrictionfragments present in the starting mixture, thereby to reduce the totalnumber of amplified fragments obtained.

As to the detection of the amplified fragment(s), the invention may beused to specifically detect one or more (oligonucleotides correspondingto) restriction fragments, which will usually correspond to one or moreAFLP® markers of interest, thus directly providing information on thepresence or absence of said marker(s) in the starting mixture.

Alternatively, or at the same time, the invention may also be used togenerate and detect a set of oligonucleotides corresponding to allrestriction fragments that were (selectively) amplified from theoriginal restriction fragment mixture, and thus to provide—forinstance—a pattern of peaks in a mass spectrum corresponding to each ofthese oligonucleotides. Such a pattern would then provide/represent akind of “fingerprint” for the starting mixture—i.e. comparable to apattern of bands obtained through gelelectrophoresis—which could beanalysed, processed further and/or stored/compiled to form a database,e.g. in a manner essentially as known for conventional geneticfingerprints.

Thus, in a further aspect, the invention relates to results and/or dataobtainable by analysing a nucleic acid or mixture of nucleic acids viathe method of the invention. These results or data may for instance bein the form of a graph, an image, a score, a set of numbers, a massspectrum, a chromatogram, digital or analogue data, or in anothersuitable form; and may optionally be stored on a suitable data carrier,including paper, photographic film, computer disc of files, a database,etc. This data may be as directly obtained from the MS equipment used,or may have been processed further, e.g. using a suitable computeralgorithm.

Therefore, according to this preferred aspect, the invention relates toa method for carrying out AFLP®, comprising steps (A) to (E) from EP 0534 858 as mentioned above, in which the amplified fragments obtained instep (D) are identified in step (E) by a mass spectroscopy technique, inparticular of MALDI-TOF; and/or a chromatography technique, such as highpressure liquid chromatography (HPLC); or a suitable combinationthereof, such as Gas Chromatography-Mass Spectroscopy (GC-MS).Preferably, a mass spectroscopy technique is used, and more preferablyMALDI-TOF.

In yet another preferred aspect, the invention relates to a method forcarrying out AFLP®, comprising steps (A) to (E) from EP 0 534 858, inwhich the amplified fragments obtained in step (D) are identified instep (E) by at least generating at least one ssDNA from at least one ofthe amplified fragments followed by identification/detection of thessDNA via a detection method based upon the length, size, mass of thessDNA. According to this aspect, the ssDNA can be identified using amass spectroscopy technique, in particular MALDI-TOF.

Alternatively, the ssDNA may be detected using another technique basedupon the nucleotide sequence of the fragment(s) to be detected, as thismay improve the resolution/resolving power. Such a sequence-baseddetection technique may (also) allow for a distinction to be madebetween oligonucleotides that have the same molecular mass (e.g. in thatthey contain the same number of each of the nucleotides A, T, G or C)but different sequences (e.g. in that the sequence of said nucleotidesA,T,G and/or C is different, in particular in that part of theoligonucleotides that is derived from the first dsDNA).

Also, in another preferred aspect, the invention relates to the use ofan adapter that contains at least one (sequence corresponding to a)recognition site for at least one restriction endonuclease of the IIStype in AFLP®; and/or to the use of a primer that contains at least one(sequence corresponding to a) recognition site for at least onerestriction endonuclease of the IIS type in AFLP®.

Yet another preferred aspect relates to the use of mass spectroscopy,and in particular of MALDI-TOF, in the detection/identification ofamplified restriction fragments generated using AFLP®.

Another preferred aspect relates to the use of a chromatographytechnique, such as high pressure liquid chromatography (HPLC); or asuitable combination of a chromatography technique and a massspectroscopy technique, such as Gas Chromatography-Mass Spectroscopy(GC-MS), in the detection/identification of amplified restrictionfragments generated AFLP®.

Preferably, in these aspects, the mass spectroscopy technique, thechromatography technique, or the combined chromatography/massspectroscopy technique is applied to ssDNA fragments, generated fromamplified dsDNA restriction fragments.

In this AFLP-based embodiment of the invention, the first dsDNA is arestriction fragment present in a mixture of restriction fragments,preferably obtained by restricting a starting DNA with at least one“frequent cutter” restriction enzyme and at least one “rare cutter”restriction enzyme, for which reference is inter alia made to EP-A-0 534858 and EP-A-721 987, both incorporated herein by reference.

The fragments thus obtained are ligated to AFLP® adapters containing anIIS recognition site. For the remainder, these adapter will beessentially equivalent to, and used in the same way as, conventionalAFLP® adapters. However, in order to provide eventually ssDNA fragmentsof a size most suitable for detection by MALDI-TOF, the adapters shouldhave a length of preferably at most 40 base pairs.

Also, the IIS recognition site should be at a position that is at leastwithin 10 base pairs of the end of the adapter that is attached to therestriction fragment, depending upon the IIS endonuclease used. Asdescribed above, this is to ensure that, when the amplified restrictionfragments are restricted with the IIS endonuclease—i.e. in step d) ofthe invention—the site at which the amplified fragment is restrictedlies that part of the amplified fragment that has been derived from theoriginal restriction fragment.

Also, when the restriction fragments are generated with a frequentcutter and a rare cutter, the adapter with the IIS recognition site ispreferably attached to the end of the restriction fragment generated bythe frequent cutter, as generally, this will eventually result in adetectable ssDNA containing two more bases derived from the restrictionfragment.

After ligation of the adapters, the mixture is amplified, i.e. usingprimers that can hybridise with (at least part of) the adapter sequencein such a way as to allow extension of the primer along the restrictionfragment (template). As mentioned, these steps are essentially carriedout according to known AFLP® protocols, for which reference is againmade to—inter alia—EP-A-0 534 858, incorporated herein by reference.

Preferably, as is known from conventional AFLP® methodology, theamplification may comprise several amplification steps. Also, preferablyat least one amplification step involves the use of selective AFLP®primers, such as +1 to +6 primers. For instance, a combination of anon-selective preamplification and a selective +2/+3 amplification maybe used, with the lowest number of selective nucleotides preferablyincluded in the primer with the recognition sequence of the IISrestriction endonuclease.

The amplified mixture of fragments thus obtained is then analysed inaccordance with the present invention, i.e. according to steps d) and e)above. For this purpose, in a first step, the amplified mixture isrestricted with the IIS endonuclease. This generally provides two typesof IIS restricted fragments, i.e. fragments comprising the adapter andpart of the original restriction fragment; and fragments comprising theremainder of the original restriction fragment (It should be noted that,as in AFLP® adapters are attached to both sides of a restrictionfragment, the invention may provide—for each amplified fragment—two IISrestricted fragments containing an adapter—each derived from one end ofthe amplified fragment—and one IIS restricted fragment comprising theremainder of the original restriction fragment). For this purpose, theAFLP-adapters may contain recognition sites of the same or different IISrestriction endonucleases, and in case of different IIS recognitionsites in the respective adapters, subsequent restriction with thecorresponding type IIS restriction endonuclease(s) may be carried outseparately of simultaneously.

The IIS-restricted fragments can then be detected. Preferably, as withthe general method set out above, this is carried out according to stepse1) and e2), i.e. by generating at least one ssDNA fragmentcorresponding to at least one of the dsDNA IIS restricted fragments.Also, this ssDNA fragment is again preferably derived from the IISrestricted fragment(s) containing the adapter sequence. Some preferred,but non-limiting methods for detecting the IIS restricted fragments, andin particular for generating the ssDNA fragments from the IIS restrictedamplified ds-fragments, will now be described hereinbelow.

Generally, detectable ssDNA fragments could be generated by separatingthe IIS restricted dsDNAs into ssDNA fragments, and then detecting allssDNAs thus obtained, or only a subset thereof (for instance generatedusing a suitable separation step). However, such a method—although notexcluded from the scope of the invention—would be cumbersome and timeconsuming, and might not provide the desired resolution.

Therefore, the ssDNA fragment to be detected is preferably generated bythe use of an exonuclease, which is used to degrade all nucleic acid(s)present in the mixture obtained after IIS restriction, except the ssDNAstrand that is eventually (to be) detected.

For this purpose, the ssDNA fragment to be detected must be resistant todegradation by the exonuclease used. This can be achieved by using, inthe amplification step c), an exonuclease-resistant primer, which duringthe amplification reaction is incorporated into (one of) the amplifiedstrand(s). In a further preferred embodiment, the exonuclease-resistantprimer also contains a biotin label, allowing for easy purification andmanipulation of the amplified fragments through its affinity forstreptavidin, whereby the latter may be coupled to a solid support suchas e.g. magnetic beads.

As the exonuclease, any exonuclease known per se can be used. Mostpreferably, a 5′→3′ exonuclease is used, such as T7 gene 6 exonucleaseand λ exonuclease. These exonucleases and their use in are well known inthe art, for instance from WO 94/16090 and EP 0 744 470.

The primer used will be resistant to the exonuclease used. In caseof—for instance—a T7 gene 6 exonuclease or a λ exonuclease, the primerwill usually contain within its sequence one and preferably severalnucleotide derivatives that make the primer resistant to theexonuclease. These may include, but are not limited to, nucleotidederivatives in which one or two of the non-bridging oxygen atoms fromthe phosphate moiety has/have been replaced by a sulphur containinggroup such as phosphorothioate group, by an alkyl group, by a nitrogencontaining group such as an amine group, or by a selenium containinggroup. The use of phosphorothioate nucleotides is especially preferred.For a further description of such exonuclease resistant primers andtheir preparation, reference is again made to WO 94/16090 and EP 6 744470.

In the amplification step c), these exonuclease resistant primers arefurther used as described above—i.e. in a manner known per se forAFLP®—upon which they are incorporated into the amplified ligated dsDNAobtained, making one strand thereof exonuclease-resistant. If severalamplification steps are used, such as a non-selective preamplificationfollowed by a selective amplification, the exonuclease-resistant primershould be used in the final amplification step.

The amplified dsDNA thus obtained is then restricted in step d) with theIIS restriction endonuclease, essentially as described above, togenerate the first and second IIS restricted dsDNAs. From this IISrestricted mixture, in step e1), an ssDNA appropriate for detection byMALDI-TOF can then be generated treatment with the appropriateexonuclease, optionally after a suitable pre-treatment of the dsDNAknown per se, such as heat-denaturation. (Such a pre-treatment isusually not required when T7 gene 6 exonuclease is used).

This aspect of the invention is schematically shown in FIG. 2, in which(9) represents the exonuclease-resistant primer and the other numeralsare essentially as described for FIG. 1. As will be clear from thediscussion of the preferred mode of the invention hereinabove, the firstdsDNA (1) will usually be a restriction fragment obtained by restrictinga starting DNA—e.g. genomic DNA or cDNA—with preferably) a frequent anda rare cutter; whereas the second dsDNA (2) will be an AFLP® adaptercontaining an IIS recognition site.

In the ligation step, the restriction fragment (1) is ligated to adapter(2), to provide a ligated dsDNA (4′). [Usually, a second AFLP®adapter—indicated as (10) in FIG. 2—will be ligated to the other end offragment (1). This second adapter (10) may or may not (also) contain anIIS restriction site, as further discussed below.]

In step c), the ligated dsDNA (4) is then amplified, using a exonucleaseresistant primer (9) that corresponds to adapter (2). [Besides theexonuclease resistant primer (9) usually also a second primer—not shownin FIG. 2—will be used that corresponds to adapter (10).]

After amplification, in step d), the amplified mixture is treated withthe appropriate IIS restriction enzyme. As with step d) in FIG. 1, thiswill provide a first IIS restricted dsDNA (6′) and a second IISrestricted dsDNA (7′).

The first IIS restricted dsDNA (6′) comprises the adapter (2) with theIIS recognition site (3) and part of (the sequence of) the originalrestriction fragment (1). Also, because of the presence of theexonuclease-resistant primer (9), one strand of the first IIS restricteddsDNA (6′) is exonuclease-resistant. The second IIS restricted dsDNA(7′) comprises the remaining part of the sequence of restrictionfragment (1), as well as the second AFLP® (10), and is not resistant todegradation by an exonuclease [unless the second primer used is alsoexonuclease resistant, for instance as discussed below].

In step e1), the mixture from IIS restriction is treated with theexonuclease. This will remove all nucleic acids in the mixture that arenot exonuclease-resistant, including the ssDNAs obtained from dsDNA(7′), the non-protected strand of dsDNA (6′), as well as (for example)remaining restriction fragments from the starting mixture that have notbeen amplified. This leaves the exonuclease resistant ssDNA (8′), whichcan then be detected.

Therefore, according to a preferred embodiment of the invention, in stepc), an exonuclease-resistant primer is used, preferably an exonucleaseresistant primer that corresponds to the second dsDNA of step b), i.e.the dsDNA containing the IIS recognition sequence. Also, according tothis preferred embodiment, in step e1) a ssDNA is generated by treatingat least (the strands of) the IIS restricted fragment that comprises (asequence corresponding to) the second dsDNA of step b) with anexonuclease. Preferably, in step e1), the entire IIS restricted mixtureobtained after step d) is treated with the exonuclease.

One further aspect of the invention therefore relates to the use inAFLP® of a primer that is resistant to an exonuclease, and in particularto a 5′→3′ exonuclease, such as a T7 gene 6 exonuclease and/or a λexonuclease. As will be clear from the above, the primer will usuallyalso contain a sequence that corresponds to a recognition site of an IISrestriction endonuclease.

In another aspect, the invention relates to a kit for use with theabove-mentioned method(s), said kit comprising (at least):

-   a restriction endonuclease of the IIS-type;-   at least one dsDNA adapter containing at least one (sequence    corresponding to a) recognition site for the restriction    endonuclease of the IIS-type;-   at least a primer adapted for use with said dsDNA adapter;    and optionally further components for kits for AFLP® known per se.

Said kit preferably also comprises an exonuclease, in particular a 5′→3′exonuclease, such as T7 gene 6 exonuclease and λ exonuclease, in whichcase at least one of the primers present in the kit will preferably beresistant to said exonuclease. The exonuclease resistant primer in thekit may preferably be a biotinylated primer, in which case the kit mayfurther comprises streptavidin coupled to a solid support, such as e.g.magnetic beads.

It should be clear to the skilled person that variations andimprovements on the above methodology are possible, without departingfrom the scope of the invention. These variations and improvements mayfor instance comprise the following:

-   The restriction fragment (1) may be ligated not to only one, but to    two adapters containing an IIS recognition sequence, i.e. such that    in FIG. 2, the “second” adapter (10) also contain an IIS recognition    sequence (not shown in FIG. 2). This IIS recognition sequence in the    second adapter (10) may be for the same IIS endonuclease, or for a    different endonuclease, as the recognition sequence in the “first”    adapter (2).-   The method of the invention is further carried out in essentially    the same manner as described above, albeit that—when the    adapters (2) and (10) contain recognition sequences for different    IIS endonucleases—the ligated dsDNA (4) is restricted in step d)    with two IIS endonucleases, i.e. simultaneously, consecutively    and/or independently. This will generate detectable fragments    derived from both ends of the original restriction fragment (1)—i.e.    comparable to fragments (6)/(6′), (7) or (8)/(8′)—which are    independent of each other and which may be detected separately or    simultaneously, thus improving the reliability and/or the resolution    of the invention. Also, detection of both ends of the fragment may    provide for an internal reference/standard for the relevant marker    during the MS detection, which may be of importance in the detection    of certain important AFLP® markers. It will also be clear that in    the embodiment of the invention where both ends of a restriction    fragment (1) are detected, usually also two exonuclease resistant    primers will be used for the amplification, each corresponding to    one of the adapters (2) and (10) used.-   In the method of FIG. 2, the primer corresponding to the second    adapter (10) may allow selective removal of the second IIS    restricted dsDNA (7). This embodiment is schematically shown in FIG.    3, in which (11) is the primer corresponding to the second adapter    (10). This primer (11) carries a functional group (12) that allows    fragment (7) to be (selectively) removed, for instance a biotin    group that allows the fragment(s) (7) to be selectively attached to    streptavidin coated beads (13). After removal of the fragments (7),    the first IIS restricted dsDNA (6) remaining in the supernatant may    then be detected, as dsDNA or as a corresponding ssDNA. It will be    clear to the skilled person that in this embodiment groups, ligands    or binding pairs other than biotin/streptavidin may also be used.-   Even more generally, the method of the invention comprises any    method in which either the first IIS restricted dsDNA    fragment(s) (6) or second IIS restricted dsDNA fragment(s) (7) are    selectively removed or isolated from the IIS restriction mixture    obtained after step d), upon which either the remaining and/or the    removed fragment(s) may be detected.-   Also, it may be possible to generate, from the first or second IIS    restricted dsDNA obtained in step d), not one but two ssDNAs, i.e.    one for/from each strand. In this embodiment, the two ssDNAs    generated from the same IIS-restricted dsDNA may be of different    length. For instance, the ssDNA corresponding to the complementary    (i.e. “antisense”) strand may be 2 base pairs shorter than the ssDNA    corresponding to the coding (i.e. “sense”) strand, and may serve as    an internal reference or standard, in particular for the detection    of specific AFLP® markers.-   Furthermore, when exonuclease-resistant primers based on    phosphorothioate bonds are used, the position(s) of these bonds may    be modulated and/or chosen such that the primers provide detectable    ssDNA fragments that are optimised for the detection window of the    mass spectroscopy technique used.-   During the amplification step(s), mismatch amplification may be    used/induced, which may cause the IIS recognition site to overlap    with the restriction site for the rare or frequent cutter. In this    way, the number of bases/nucleotides derived from the restriction    fragment—which determine the differences in mass or sequence that is    (to be) detected—may be increased, which may further improve the    resolution that can be obtained.-   Optionally, after the IIS restriction step d), the ds DNA    fragments—e.g. dsDNA fragments (6) and/or (7)—may be further    restricted with one or more further restriction endonucleases, such    as a combination of several “4-cutters”, to provide (a set of)    specific, detectable fragments of different length (and thereby of    different mass). This may increase the resolution and/or increase    the number of “unique” detectable fragments obtained.-   Also, according to a further embodiment of the invention, the    IIS-restriction step d) may be replaced by restriction with one or    more further restriction endonucleases, such as a combination of one    or more “4-cutters”. This may provide a set of restriction    fragments, of which one, several or essentially all fragments may    then be detected in a suitable manner, for instance based upon    differences in mass, size, weight, or sequence of the fragment(s).    Again, this may increase the resolution and/or increase the number    of “unique” detectable fragments obtained.

As mentioned above, the method of the invention can be used to analyse amixture of amplified restriction fragments generated using AFLP®. Assuch, the method of the invention can be used for all purposes thatconventional AFLP® (i.e. involving gel electrophoresis/autoradiography)and/or variants or improvements thereof can be used.

For instance, the method of the invention can be used to analyse anykind of nucleic acid sequence or mixture of nucleic acid sequences,including, but not limited to, plant-derived sequences, animal-derivedsequences, human-derived sequences, microbial sequences, yeastsequences, sequences from fungi and algae, viral sequences, as well assynthetic sequences.

For example, the method of the invention may be used to analyse DNAsequences, including genomic DNA, cDNA, structural genes, regulatorysequences and/or parts thereof; as well as RNA, including mRNA,optionally by analogous modification of the method given above. Oneparticular application of interest could be the detection of cDNA-AFLP®fragments.

In these and other applications, the method of the invention can be usedfor any purpose for which a polymorphic marker or a transcribed genesequence can be used and/or identified. This includes, but is notlimited to, all the uses described in the art for polymorphic markers inknown DNA-fingerprinting, genotyping, transcript profiling andDNA-identification techniques. The method of the invention is of courseespecially suited in these applications for which an AFLP®-marker can beused and/or identified, including those mentioned above and in EP-A-0534 858 and the co-pending European applications 98.202.5496 and98.202.4515. It is also envisaged that by the method of the invention,new genetic markers may be identified, and these form a further aspectof the invention.

For instance, the method of the invention may be used to classify anindividual as belonging to a certain species, subspecies, variety,cultivar, race, strain or line, or to study the inheritance of a genetictrait or property. Also, the method of the invention may be used todetect markers indicative of the presence, the absence or the state of agenetically determined or genetically influenced disease or disorder,including cancer, oncogenes and oncogenic mutations, in which case themethod of the invention may be used for diagnostic purposes.

Possible fields of use therefore include, but are not limited to, plantand animal breeding, variety or cultivar identification, diagnosticmedicine, disease diagnosis in plants and animals, identification ofgenetically inherited diseases in humans, family relationship analysis,forensic science, organ-transplant, microbial and viral typing such asmultiplex testing for strains of infectious diseases; as well as thestudy of genetic inheritance, gene expression, mutations, oncogenesand/or drug resistance; or for mRNA detection.

In particular, the method of the invention may be used for the highthroughput detection of single nucleotide polymorphism's or SNPs. It isexpected that on the basis of such SNPs, using association studies,important genes involved in diseases in humans and/or other organismsmay be identified and/or studied. The high sensitivity of the method ofthe invention would make it possible to distinguish between SNP alleles.

Also, the method of the invention may be used to determinequantitatively the amount of a specific DNA, DNA-fragment or markerpresent in a starting sample. For instance, in transcript profilingusing cDNA-AFLP®, the method of the invention may be used for thequantitative determination of the amount of cDNA in a biological sample.Also, the method of the invention may be used to provide quantitativedata in homozygote/heterozygote-AFLP® techniques.

Besides the advantages already mentioned hereinabove, some furtheradvantages of the method of the invention include:

-   as the method of the invention is based upon detection of the mass    or the sequence of the fragments generated, there is no interference    from cross-hybridisation effects, as may occur with    hybridisation-based detection methods including array-based    techniques.-   the method of the invention may make it possible for specific    markers to be determined or scored quantitatively, e.g. based on    peak area and/or peak height.-   the use of radioactive or fluorescent labels is not required    (although a suitable use thereof is not excluded from the scope of    the invention).-   the use of mass labels is not required (although a suitable use    thereof is not excluded from the scope of the invention).-   in most of its embodiments, the invention only comprises enzymatic    steps. These can be carried out in a single reaction vessel, and/or    can easily be automated.-   Contrary to gel-based detection techniques, AFLP®-markers of all    sizes can be detected. These include fragments/markers of sizes that    cannot be detected easily using current gel-electrophoresis    techniques, e.g. fragments/markers of 600 base pairs or more.-   the method of the invention can achieve high throughput. Also,    results for a specific sample can be generated quickly.-   In principle, the presence or absence of a single peak in the mass    spectrum or chromatogram generated may already be indicative for the    presence of absence of a marker or cDNA-AFLP fragment of interest.

In another embodiment of the invention, schematically shown in FIG. 6,oligonucleotides suitable for detection using MALDI-TOF and/or one ofthe other techniques mentioned above are generated using a restrictionendonuclease that is capable of restricting a starting nucleic acid(indicated as (14) in FIG. 6) at two sites, in particular at two sitesdifferent from the recognition site, and more in particular one sitelocated upstream of the recognised sequence(s) and one site locateddownstream of the recognised sequence(s). (In FIG. 6, the recognitionsite is indicated as (15) and the two restricted sites (are indicated as(16) and (17), respectively).

Some non-limiting examples of such “double cutting” restrictionendonucleases are: BaeI, which has the recognition sequence:

-   BaeI, which has the recognition sequence:

N N N N N{circumflex over ( )}N N N N N N N N N N A C N N N N G T A Y CN N N N N N N N N N N N{circumflex over ()} (SEQ ID NO.59) {circumflexover ()}N N N N N N N N N N N N N N N T G N N N N C A T R G N N N N N NN{circumflex over ( )}N N N N N (SEQ ID NO.60)

-   BcgI, which has the recognition sequence:

N N{circumflex over ( )}N N N N N N N N N N C G A N N N N N N T G C N NN N N N N N N N N N{circumflex over ()} (SEQ ID NO.61) {circumflex over( )}N N N N N N N N N N N N G C T N N N N N N A C G N N N N N N N N NN{circumflex over ( )}N N (SEQ ID NO.62)

-   BplI, which has the recognition sequence:

N N N N N{circumflex over ( )}N N N N N N N N G A G N N N N N C T C N NN N N N N N N N N N N{circumflex over ()} (SEQ ID NO.63) {circumflexover ( )}N N N N N N N N N N N N N C T C N N N N N G A G N N N N N N NN{circumflex over ( )}N N N N N (SEQ ID NO.64)

-   Bsp24I, which has the recognition sequence:

N N N N N{circumflex over ( )}N N N N N N N N G A C N N N N N N T G G NN N N N N N N N N N N{circumflex over ()} (SEQ ID NO.65) {circumflexover ( )}N N N N N N N N N N N N N C T G N N N N N N A C C N N N N N NN{circumflex over ( )}N N N N N (SEQ ID NO.66)

-   CjeI, which has the recognition sequence:

N N N N N N{circumflex over ( )}N N N N N N N N C C A N N N N N N G T NN N N N N N N N N N N N N N{circumflex over ()} (SEQ ID NO.67){circumflex over ( )}N N N N N N N N N N N N N N G G T N N N N N N C A NN N N N N N N N{circumflex over ( )}N N N N N N (SEQ ID NO.68)

-   CjePI, which has the recognition sequence:

N N N N N N{circumflex over ( )}N N N N N N C C A N N N N N N N T C N NN N N N N N N N N N N N{circumflex over ()} (SEQ ID NO.69) {circumflexover ( )}N N N N N N N N N N N N G G T N N N N N N N A G N N N N N N NN{circumflex over ( )}N N N N N N (SEQ ID NO.70)and AloI and Hin4I, of which BcgI is preferred, due to the fact thatonly two base overhangs are created, which improved the specificity ofligating the second dsDNA to the first dsDNA.

Thus, by restricting a starting nucleic acid, such as a dsDNA, inparticular a genomic DNA or a cDNA, with such a “double cutting”restriction endonuclease, it is possible to generate one or morefragments of between 20–50, usually between 30–40 b.p., and often 34 or36 bp in length/size. These fragments, which are indicated as (18) inFIG. 6, usually will contain the recognition site/sequence(s) (10) ofthe restriction endonuclease used, as well as about 30 (depending on therestriction endonuclease used) “unknown” bases which are fully dependantupon the starting nucleic acid used. Thus, besides having a sizesuitable for detecting by MALDI-TOF and/or one of the other techniquesmentioned above, these fragments are also specific/representative forthe starting nucleic acid (14).

Usually, when a plant-derived DNA is used as the starting nucleic acid(14), such “double cutting” restriction endonucleases will cut said DNAwith a frequency of about once per 3000 base pairs. Thus, this generallyprovides a mixture of the “small” restriction fragments (18) mentionedabove and “large” restriction fragments. These large fragments, whichare indicated as (19) in FIG. 6, will usually have a size of severalhundreds or even thousands of base pairs, and on average about 2000 to3000 base pairs, and usually will not contain a sequence correspondingto the recognition site/sequence(s) of the restriction endonucleaseused.

In principle, it could be possible to specifically detect one or more—oressentially all—of the fragments(18) and/or (19) present in the thusrestricted mixture, and in particular to detect one or more—oressentially all—of the small fragments (18), optionally after isolatingsaid fragment(s) (18) from the restricted mixture and/or selectivelyremoving the large fragments (19).

According to the invention, however, the restricted mixture is mostpreferably first amplified, even more preferably using at least oneselective amplification step in order to reduce the complexity of thefragment mixture. Then, according to this preferred method, one ormore—or essentially all—of the amplified fragments (18) and /or (19),and in particular one or more of the amplified small fragments (18), aredetected, e.g. using MALDI-TOF and/or one of the other detectiontechniques mentioned above.

Said amplification is most preferably carried out using known AFLP®methodology, i.e. by ligating the fragments present in the restrictedmixture—i.e. both the small fragments (18) as well as the largefragments (19)—to at least one adapter and then amplifying the mixturesusing primers suitable for use with said adapters. (In FIG. 6, theadapters are indicated as (20) and the primers are indicated as (21).This can be carried out essentially a described hereinabove and in—interalia—EP-A-0 534 858, incorporated herein by reference.

Also, said amplification may comprise a single amplification step orseveral amplification steps. As already mentioned, in order to reducethe complexity of the mixture, in (at least one of) the amplificationstep(s), a primer containing at least one selective nucleotide is used,as is known from AFLP® methodology. For instance, a combination of anon-selective preamplification and one or more selective amplificationsmay be used, or a combination of several selective amplification steps,each using primers with an increasing number of selective nucleotides,e.g. as described in Example III below. Preferably, in order to avoidmismatches, in an amplification comprising several amplification steps,the number of selective nucleotides is not increased by more than 1 or 2selective nucleotides for each round of amplification.

Also, in the amplification preferably a mixture of at least two adaptersis used, more preferably adapters with differing adapter-core sequences(not shown FIG. 6). With these adapters, most preferably also a mixtureof at least two primers suitable for use with these said adapters areused (also not shown in FIG. 6).

Also, preferably, the amplification conditions are chosen such that theadapter-ligated “small” fragments (18) are amplified more efficientlythen the adapter-ligated “large” fragments (19), e.g. by using aamplification profile (e.g. time, temperature) that is sufficient toallow for full elongation of the primer(s) (21) along theadapter-ligated short fragments (18) but that is insufficient for fullelongation of the primers(s) (21) along the adapter-ligated largefragments (19).

After the amplification, an amplified mixture is obtained that for themajor part consists of the amplified adapter-ligated “small” fragments(indicated as (22) in FIG. 6) and the amplified adapter-ligated “large”fragments (indicated as (23) in FIG. 6). This amplified mixture can thenbe analysed in a manner known per se, i.e. by (specifically) detectingone or more—or essentially all—of the amplified adapter-ligated “small”fragments (22), one or more—or essentially all—of the amplifiedadapter-ligated “large” fragments (23), or a combination thereof,optionally, after removing the adapter sequences by digestion with the“double cutting” restriction enzyme to improve the resolution ofdetection.

Preferably, one or more—or essentially all—of the amplifiedadapter-ligated “small” fragments (22) are detected, e.g. using massspectroscopy such as MALDI-TOF, and/or one of the other techniquesmentioned above, such as a chromatography technique; or a suitablecombination thereof. For this purpose, the length of the adapters(20)/primers (21) used in the amplification step(s) is preferably chosensuch that the amplified adapter-ligated “small” fragments (22) obtainedare still of a size suitable for such detection, e.g. as mentionedabove. When conventional AFLP-adapters and -primers are used, e.g. ofabout 10–30 base pairs in length, this will usually be the case, e.g.provide amplified adapter-ligated “small” fragments (22) of between 50and 100 bp, preferably between 60 ad 80 bp, and usually about 70 bp.

Again, as in the methods described above, the one or moreadapter-ligated small fragments (22) may be detected as double strandedfragments (e.g. as directly obtained after amplification), or as singlestranded sequences generated from the amplified double strandedfragments (22) (not shown in FIG. 6).

Before detection, the one or more adapter-ligated small fragments (22)to be detected may be (specifically) isolated from the amplifiedmixture. For instance, the one or more adapter-ligated small fragments(22) may be separated from the adapter-ligated “large” fragment(s) (23)and/or any other fragments/components present in the amplified mixture,or the adapter-ligated “large” fragment(s) (23) may be (selectively)removed from the amplified mixture.

This may be carried out in any suitable manner, for instance using asuitable separation technique, such as a technique based on thedifference(s) in size between the small fragments (22) and the largefragments (23). Alternatively, primers (22) may be used in theamplification that allow for (selective) isolation of the amplifiedfragments. For instance, in the final amplification step, a primer (21)may be used that carries a biotin group, which allows the amplifiedfragments to be selectively isolated using a streptavidin-coatedcarrier, such as streptavidin coated magnetic beads (not shown in FIG.6).

Also, optionally, in the (final) amplification (step), a set of primersmay be used that each have different mass. This would allow analysis ofdifferent primer combinations simultaneously without overlap between themasses of the different fragments/oligonucleotides.

This aspect of the invention generally provides the same advantages asalready mentioned above. In particular, a short time is required fordetection, which allows for high throughput. Also, there is no need touse autoradiography and/or radioactive/fluorescent labels, andoligonucleotides are obtained that are specific for the starting nucleicacid and that can be analysed using a detection technique based on(differences in) the mass.

Furthermore, compared to the method described above, this aspect of theinvention offers the following further advantages:

-   A higher proportion—i.e. essentially all in case of digestion of the    amplified ligated restriction fragments with the “double cutting    RE”—of the nucleotides in the amplified (small) fragments (18) that    are eventually detected—e.g. as amplified fragments (22)—will be    derived from the starting nucleic acid (14). These generally include    the nucleotides that correspond to the recognition sequence (17) of    the “double cutting” restriction endonuclease used, as well as the    about 30 “unknown” nucleotides present in the small fragments (18).    This not only means the differences in mass between the individual    oligonucleotides (22) to be detected are increased, but also that    the chances that different fragments (18)/(22) will have the same    mass (but for instance only a different nucleotide sequence) will be    reduced, e.g. to 3% or less. This improves sensitivity and/or    resolution.-   The primers used in the amplification will be independent of the    restriction enzyme used.

Thus, this aspect of the invention relates to a method for generating,and optionally detecting, an oligonucleotide, comprising at least thesteps of:

-   a) providing a dsDNA;-   b) restricting the dsDNA with at least one restriction endonuclease    that restricts the dsDNA at two sites different from the recognition    site of said restriction endonuclease, so as to provide a mixture of    restricted fragmented, said mixture comprising one or more fragments    that contain the recognition site/sequence(s) of the restriction    endonuclease and one or more fragments that do not contain a    sequence corresponding to the recognition site/sequence(s) of the    restriction endonuclease used; and-   d) detecting at least one of the restricted fragments obtained in    step b).

In step (d), preferably one or more—or essentially all—of the fragmentsthat contain the recognition site/sequence(s) of the restrictionendonuclease are detected, optionally after (specific) isolation ofthese (one or more) fragments from the mixture obtained in step b).

Preferably, this method comprises the steps of:

-   a) providing a dsDNA;-   b) restricting the dsDNA with at least one restriction endonuclease    that restricts the dsDNA at two sites different from the recognition    site of said restriction endonuclease, so as to provide a mixture of    restricted fragments, said mixture comprising one or more fragments    that contain the recognition site/sequence(s) of the restriction    endonuclease and one or more fragments that do not contain a    sequence corresponding to the recognition site/sequence(s) of the    restriction endonuclease used;-   c) amplifying the mixture of fragments obtained in step b);-   d) detecting at least one of the amplified fragments obtained in    step c), or, optionally, after digesting the amplified fragments    with the “double cutting RE” to remove the adapter sequences.

In step (d), preferably one or more—or essentially all—of the amplifiedfragments that contain the recognition site/sequence(s) of therestriction endonuclease are detected, optionally after specificisolation of these (one or more) fragments from the amplified mixtureobtained in step c). Preferably, the primer(s) used in the amplificationin step (c) are biotinylated, such that the fragments from the amplifiedmixture may be isolated using streptavidin on a solid support such ase.g. magnetic beads.

The amplification of step c) is preferably carried out using AFLP®methodology, e.g. as described above. The detection of step d) ispreferably carried out using mass spectroscopy such as MALDI-TOF, or achromatography technique, such as high pressure liquid chromatography(HPLC); or a suitable combination of a chromatography technique and amass spectroscopy technique, such as Gas Chromatography-MassSpectroscopy (GC-MS).

For further preferences and embodiments of this aspect of the invention,reference is made to the general description above.

The invention will now be illustrated by means of the followingnon-limiting Experimental Part.

Experimental Part

EXAMPLE I

AFLP fingerprints produced with AFLP adapters and selectiveamplification primers, which contain a recognition site for type IISrestriction enzymes GsuI or BsgI.

AFLP fingerprints generated with the standard AFLP adapters- andselective amplification primers, which do not contain a type IIS site,are included as controls.

1. Biological Material

The biological materials used are the parental lines of a population ofrecombinant inbred lines of Arabidopsis. These parental lines are theecotypes Colombia (sample No. NW20) and Landsberg erecta (sample nr.N933) and were acquired from the arabidopsis stock centre in Nottingham(UK).

2. EcoRI/MseI AFLP Template Preparation, Preamplification and SelectiveAmplification.

All procedures were conducted according to the standard protocols (Voset al., Nucleic Acids Research 23: no 21, pp. 4407–4414, and patentapplication EP0534858). AFLP reactions were labelled radioactively with³³P and resolved on a standard AFLP (sequence) gel. The sequences of theadapters, preamplification primers and selective amplification primersare as follows:

2.1 Standard AFLP Procedure (FIG. 4, Lanes 1 and 2):

EcoRI adapter: 91M35: 5′-ctcgtagactgcgtacc-3′ and (SEQ ID NO.1) 91M36:3′-catctgacgcatggttaa-5′ (SEQ ID NO.2) EcoRI + 1 preamplication primer:E01K: 5′-gactgcgtaccaattca-3′ (SEQ ID NO.3) EcoRI + 2 selectiveamplification primer: E12: 5′-gacctgcgtaccaattcac-3′ (SEQ ID NO.4) MseIadapter: 92A18: 5′-gacgatgagtcctgag-3′ (SEQ ID NO.5) 92A19:3′-tactcaggactcat-5′ (SEQ ID NO.6) MseI + 1 preamplification primer:M02: 5′-gatgagtcctgagtaac-3′ (SEQ ID NO.7) MseI + 3 selectiveamplification primer: M47: 5′-gatgagtcctgagtaacaa-3′ (SEQ ID NO.8)2.2 AFLP Fingerprints Generated with MseI Adapter- and AFLP PrimersContaining a GsuI Site (5′-3′ CTGGAG N₁₆/N₁₄)(SEQ ID NO.71). (FIG. 4Lanes 3 and 4).

EcoRI adapter, preamplification AFLP primer and selective AFLP primer:for sequences see 2.1.

MseI adapter: 99I21: 5′-gacgatgagtctggag-3′ (SEQ ID NO.9) 99I22:5′ tactccagactcat-3′ (SEQ ID NO.10) MseI + 1 preamplication primer:99I23: 5′-gacgatgagtctggagtaac-3′ (SEQ ID NO.11) MseI + 3 selectiveamplification primer: 99I25: 5′-gacGATGAgtctggagtaacaa-3′ (SEQ ID NO.12)(nucleotides shown in uppercase are joined by phosphorothioate bonds toconfer resistance to T7 gene 6 exonuclease).2.3 AFLP Fingerprints Generated with MseI Adapter- and AFLP PrimersContaining a BsgI Site (5′-3′ GTGCAG N₁₆/N₁₄)(SEQ ID NO.15). (FIG. 4Lanes 5 and 6).

EcoRI adapter, preamplification AFLP primer and selective AFLP primer:for sequences see 2.1.

MseI adapter: 99I37: 5′-gacgatgagtgtgcag-3′ (SEQ ID NO.13) 99I38:5′-tactgcacactcat-3′ (SEQ ID NO.14) MseI + 1 preamplification primer:99I40: 5′-tactgcacactcat-3′ (SEQ ID NO.14))MseI+3 selective amplification primer:99I41: 5′-gacGATCAgtgtgcagtaacaa-3′ (SEQ ID NO.16)(nucleotides shown in uppercase are joined by phosphorothioate bonds toconfer resistance to T7 gene 6 exonuclease).3. Result

FIG. 4 shows that very similar fingerprints are obtained when thestandard AFLP adapters and primers are used (lanes 1 and 2) or when AFLPadapters and primers are used which contain a GsuI site (lanes 3 and 4)or a BsgI type IIS site (lanes 5 and 6).

-   Lanes 1, 3 and 5 contain the EcoRI/MseI +2/+3 fingerprints derived    from sample N933.-   Lanes 2, 4 and 6 contain the EcoRI/MseI +2/+3 fingerprints derived    from sample NW20.-   (the +2/+3 selective nucleotides are the same in all cases (lanes    1–6) and correspond to EcoRI primers E12 (+AC) and MseI primer M47    (+CAA).-   Lanes 7 contains a 10 basepair ladder a size reference.

EXAMPLE II Restriction Enzyme Digestion of AFLP Reactions Generated withan MseI AFLP Adapter and Amplification Primer Containing a Type IISRestriction Enzyme Site.

1. Biological Material

The biological material used are two recombinant inbred lines (RIL) ofArabidopsis (samples numbers 1923 and 1904). The parental lines are ofthe RIL population are the ecotypes Colombia and Landsberg erecta. TheRIL were acquired from the arabidopsis stock centre in Nottingham (UK).

2. EcoRI/MseI AFLP Template Preparation, Preamplification and SelectiveAmplification.

All procedures were conducted according to the standard protocols (Voset al., Nucleic Acids Research 23: no 21, pp. 4407–4414, and patentapplication EP0534858. The sequences of the adapters, preamplificationprimers and selective amplification primers are as described in ExampleI with the exception of:

2.1 Standard AFLP Amplification:

Selective amplification primer M47 was substituted for primer

MS50: 5′-gatgagtcctgagtaacat-3 (SEQ ID NO.17)

2.2 AFLP Fingerprints Generated with MseI Adapter- and AFLP PrimersContaining a GsuI Site.

+3 MseI selective amplification primer I25 was substituted for primerI32: 5′-gacgATGAGtctggagtaacag-3′ (SEQ ID NO.18)

(nucleotides shown in uppercase are joined by phosphorothioate bonds toconfer resistance to Exonuclease T7 gene 6).

2.3 AFLP Fingerprints Generated with MseI Adapter- and AFLP PrimersContaining a BsgI Site.

+3 MseI selective amplification primer I41 was substituted for primerI48: 5′-gacgATGAGtgtgcagtaacat-3′ (SEQ ID NO.19)

(nucleotides shown in lowercase are joined by phosphorothioate bonds toconfer resistance to T7 gene 6 exonuclease).

3. Preparing AFLP Reactions with Double Stranded Fragments.

After the selective amplification reaction according to the standardprocedure in a 20 microliter volume with primers sequences are describedin sections 2.1, 2.2 and 2.3, an identical amount of 20 microliterselective amplification reagents was added to yield a 40 microlitertotal volume, and an extra cycle of PCR amplification was conductedaccording to the thermal cycle profile 30 sec 94° C., 30 sec 56° C. and4 min 72° C., to convert all AFLP fragments to double stranded DNA.

4. Restriction Enzyme Digestion.

Following the preparation of double stranded AFLP reactions as describedin sections 2 and 3 above, a 5 microliter sample was taken from eachtube as control prior to restriction enzyme digestion. To the remainderof each tube (35 microliter volume), 15 microliter restriction ligationbuffer was added to yield a 50 microliter total volume, and 5microliters GSuI or BsgI restriction enzyme was added to AFLP reactionsprepared with the corresponding AFLP adapter- and primer sequences. Thesamples were incubated for 110 minutes at 37° C. (BsgI) or 30° C.(GsuI). After restriction enzyme digestion another 5 microliter aliquotwas taken, mixed with loading dye, and loaded along with the undigestedcontrol samples on a 18% polyacrylamide gel.

5. Results

FIG. 5 contains:

AFLP reactions prepared with GsuI site containing MseI adapter- and AFLPprimers:

-   Lane 1: sample 1923, AFLP reaction E12/I32 (GsuI site) before    restriction enzyme digestion.-   Lane 2: sample 1904, AFLP reaction E12/I32 (GsuI site) before    restriction enzyme digestion.-   Lane 3: sample 1923, AFLP reaction E12/I32 (GsuI site) after    restriction enzyme digestion.-   Lane 4: sample 1904, AFLP reaction E12/I32 (GsuI site) after    restriction enzyme digestion.    AFLP reactions prepared with BsgI site containing MseI adapter- and    AFLP primers:-   Lane 5: sample 1923, AFLP reaction E12/I48 (BsgI site) before    restriction enzyme digestion.-   Lane 6: sample 1904, AFLP reaction E12/I48 (BsgI site) before    restriction enzyme digestion.-   Lane 7: sample 1923, AFLP reaction E12/I48 (BsgI site) after    restriction enzyme digestion.-   Lane 8: sample 1904, AFLP reaction E12/I48 (BsgI site) after    restriction enzyme digestion.    Standard AFLP reactions-   Lane 9: sample 1923, standard AFLP reaction E12/M50 before    restriction enzyme digestion.-   Lane 10: sample 1904, standard AFLP reaction E12/M50 before    restriction enzyme digestion.-   Lane 11: sample 1923, standard AFLP reaction E12/M50 after    restriction enzyme digestion.-   Lane 12: sample 1904, standard AFLP reaction E12/M50 after    restriction enzyme digestion.    Size ladder-   Lane 13: contains a 10 basepair size ladder as a reference for    fragment size.    FIG. 5 shows:-   1) A strong fragment of 32 bases in lanes 3 and 4 as expected after    digestion with GsuI.-   2) A strong fragment of 32 bases in lanes 7 and 8 as expected after    digestion with BsgI.-   3) No restriction fragment of 32 bases in the remaining lanes    (samples prior to digestion with GsuI or BsgI or any of lanes 9–12    which contain the standard AFLP reactions).    In conclusion FIG. 5 shows that double stranded AFLP oligotags can    be generated by preparation of AFLP templates with adapters and AFLP    primers containing a BsgI or GsuI site, and digestion of AFLP    reactions containing double stranded DNA fragments with these    enzymes.

EXAMPLE III Generating Specific Detectable Oligonucleotides Using a“Double Cutting” Restriction Endonuclease.

Genomic DNA is digested using BcgI, to provide a mixture that containssmall fragments with the BcgI recognition sequence(s), as well as largefragments that do not contain any BcgI recognition site. The fragmentscontaining BcgI recognition site can be schematically represented asfollows:

5′ nnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnnnn-3′ (SEQ ID NO. 72)3′ nnnnnnnnnnnnGCTnnnnnnAGCnnnnnnnnnn-5′ (SEQ ID NO. 73)

The mixture of fragments is ligated to two different adapters (adaptersX: xxxxxNN, adapter Y: yyyyyNN), each with a different core sequence,and amplified. The bottom strand will not ligate, but will be “filledin” during the first amplification step. This provides fragments havingon their respective termini the adapters X-X, X-Y, Y-X or Y-Y.

x x x x x NN nnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnn n n (SEQ ID NO. 74)YYYYYXXXXX n n nnnnnnnnnnGCTnnnnnnACGnnnnnnnnnn N N y y y y y (SEQ IDNO. 75)

The complexity of the mixture is reduced through amplification withprimers containing selective nucleotides. To prevent mismatching,amplifications are carried out in which the number of selectivenucleotides used in each amplification step is not increased by morethan 1 or 2 per amplification step. Only the fragments with differentadapters on both termini will amplify exponentially. Optionally, for afinal +ATG/+CGA extension 2 or 3 consecutive amplifications are carriedout. In the final amplification step, one primer will be labelled with abiotin-molecule (“bio”).

bio- x x x x xATGnnnnnnnnnCGAnnnnnnTCGnnnnnnnnnTCG (SEQ ID NO. 76)YYYYYXXXXXTACnnnnnnnnnGCTnnnnnnACGnnnnnnnnnACG y y y y y (SEQ ID NO. 77)

The fragments are purified using streptavidin beads and theoligonucleotide-tags are isolated by treatment of the beads with alkalior by heat denaturation. The tags are obtained in the supernatant of thealkali or heat treatment.

-   XXXXXTACnnnnnnnnnGCTnnnnnnACGnnnnnnnnnAGCYYYYY(SEQ ID NO. 78)

The oligonucleotide tags are analysed using mass spectroscopy, e.g. bydetermining the mass of the tags with a length of 70 nucleotides,optionally, after digestion with BcgI to remove the non-informativeadapter sequences, which results in a higher resolution to detect theresulting tags with a length of 32 nucleotides.

This method may be carried out as follows;

DNA-template is prepared by digesting 200–500 ng genomic DNA with 5units of BcgI. The restriction fragments thus obtained are ligated to amixture of the following adapters:

Adapter 1:  (SEQ ID NO.20 and 21) 5′ CTCGTAGACTGCGTACCNN 3′3′ GAGCATCTGACGCATGG 5′ Adapter 2:  (SEQ ID NO.22 and 23)5′ GACGATGAGTCCTGAGANN 3′ 3′ CTGCTACTCAGGACTCT 5′The mixture of adapter-ligated fragments is then amplified using apre-amplification with a set of +1 and +2 primers for use with adapter 1and adapter 2, respectively i.e.:

+1 primer for Adapter 1: CTCGTAGACTGCGTACCN (SEQ ID NO.24) +2 primer forAdapter 2: GACGATGAGTCCTGAGACN (SEQ ID NO.25)

The preamplification profile comprises a short denaturation (1 sec, 94°C.) followed by a short elongation (5 sec., 56° C.), both repeated 40times. Such a profile favours amplification of the adapter-ligated smallfragments compared to amplification of the adapter-ligated largefragments.

The +1/+2 preamplification is followed by a selective +2 amplification,selective +3 amplification, or a selective +4 amplification (or acombination of +2, +3, or +4 selective amplifications) using thefollowing primers:

+2 primer for Adapter 1: CTCGTAGACTGCGTACCNN (SEQ ID NO.26) +3 primerfor Adapter 2: GACGATGAGTCCTGAGACNN (SEQ ID NO.27) +3 primer for Adapter1: CTCGTAGACTGCGTACCNNN (SEQ ID NO.28) +4 primer for Adapter 2:GACGATGAGTCCTGAGACNNN (SEQ ID NO.29)

One of the primers used in the final amplification step contains a5′-biotin group. The mixture of amplified fragments is then isolated andpurified by contacting the mixture with streptavidin coated magneticbeads. The oligonucleotide fragments are isolated by incubation of thebeads carrying the restriction fragments with alkali or by heating (5min. 95° C.). The supernatant thus obtained, containing the amplifiedrestriction fragments, may then be analysed, e.g. using a massspectroscopy technique such as MALDI-TOF. The selective amplificationprofile comprises a short denaturation (1 sec. 94° C.), followed by ashort elongation (5 sec. 65° C.), both repeated 13 times, in which theelongation temperature is gradually lowered to 56° C. by a decrease of0.7° C. per cycle, followed by another 23 cycles consisting of adenaturation step of 1 sec. at 94° C. and an elongation step of 5 sec.at 56° C.

EXAMPLE IV

Generation of Oligotags from AFLP Reactions Generated with a MseI AFLPAdapter and an Amplification Primer Containing a Type IIS RestrictionEnzyme Site.

1. Biological Material

The biological material used are the two parental lines (samples IR20and 6383) from a F2 population of Rice. The parental lines were acquiredfrom the IRRI in the Philippines.

2. EcoRI/MseI AFLP Template Preparation, Preamplification and SelectiveAmplification.

All procedures were conducted according to the standard protocols (Voset al., Nucleic Acids Research 23: no 21, pp. 4407–4414 and patentapplication EP0534858). AFLP reactions were radioactively labelled with33P and resolved on a standard AFLP (sequence gel). The sequences of theadapters, preamplification primers and selective amplification primersare as described in Example I with the exception of:

2.1 AFLP Fingerprints Generated with MseI Adapter- and AFLP PrimersContaining a GsuI Site (5′-3′ CTGGAG N16/N14) (SEQ ID NO.79). (FIG. 7).

+3 MseI selective amplification primer I25 was substituted for primer00s45thio: 5′-gatgaGTCTGGAGTAACAC-3′ (SEQ ID NO.30), (nucleotides shownin lowercase are joined by phosphorothioate bonds to confer resistanceto T7 gene 6 Exonuclease).

2.2 AFLP Fingerprints Generated with MseI Adapter- and AFLP PrimersContaining a BsgI Site. (5′-3′ GTGCAG N16/N14) (SEQ ID NO.80). (FIG. 8).

+3 MseI selective amplification primer I41 was substituted for primer00s24bio: 5′-GA(bioT)GAGTGTGCAGTAACAC-3′ (SEQ ID NO.31), (bioT refers toa biotin molecule coupled to the deoxythymidinenucleoside).

3. Preparing AFLP Reactions with Double Stranded Fragments.

After the selective amplification reaction according to the standardprocedure in a 20 microliter volume with primer sequences described insections 2.1 and 2.2, an identical amount of 20 microliter selectiveamplification reagents was added to yield a 40 microliter total volume,and an extra cycle of PCR amplification was conducted according to thethermal cycle profile 30 sec 94° C., 1 min 56° C. and 2 min 72° C., intoconvert all AFLP fragments to double stranded DNA.

4. Preparation of AFLP Oligonucleotide Tags.

Following the preparation of double stranded AFLP reactions as describedin sections 2 and 3 above, a 10 microliter sample was taken from eachtube and mixed with 10 microliter loading dye as a control prior to AFLPoligonucleotide tag preparation. To 10 microliter of each tube a 5microliter mixture containing 0.5 microliter of 10× PCR buffer, 0.66units ExonucleaseI, 0.594 microliter 10× Shrimp Alkaline Phosphatasebuffer and ddH2O, was added to yield a 15 microliter total volume. Thesamples were incubated for 30 minutes at 37° C. followed by incubationfor 10 minutes at 70° C. to inactivate the Exonuclease I. To this 15microliter sample, 10 microliter restriction enzyme mix was addedcontaining 2.5 microliter restriction enzyme buffer, 3 units of GsuI orBsgI restriction enzyme and S-adenosylmethionine according to themanufacturers specifications. The samples were incubated for 180 minutesat 30° C. (GsuI) or 37° C. (BsgI). After the restriction enzymedigestion a 5 microliter aliquot was taken, mixed with loading dye, andloaded on a sequence gel along with the untreated control sample, as acontrol for the digestion by the restriction enzymes.

The remainder of the restriction enzyme digestion mix was used toisolate the oligonucleotide tags.

To the GsuI digestion mix 5 microliter 5× T7 Gene 6 Exonuclease bufferand 5 units T7 Gene 6 Exonuclease was added and incubated for 1 hour at37° C. followed by a 10 minutes incubation at 70° C. A 5 microlitersample was taken and mixed with 5 microliter loading dye and loaded on asequence gel along with the earlier mentioned samples. The remainder ofthe sample was purified using a nucleotide removal kit from Qiagen.

The sample was eluted from the Qiagen column with 100 microliter ddH2Oand concentrated by evaporation of water to an end volume of 15microliter. This 15 microliter sample was mixed with 15 microliterloading dye and loaded on sequence gel together with the earliermentioned samples.

To the BsgI digestion mix a mixture containing 5 microliter magneticstreptavidin coated beads reconstituted 80 microliter STEX solution(1000 mM NaCl/10 mM Tris HCl/1 mM EDTA/0.1% Triton X-100 pH 8.0), wasadded and incubated for 30 minutes at room temperature with gentleagitation. After this incubation the beads were concentrated with themagnetic particle concentrator and washed sequentially twice with 100microliter STEX and once with 100 microliter ddH2O and reconstituted in15 microliter. The oligonucleotide tags were incubated for 5 minutes at95° C. and after concentration on a magnetic particle concentrator thesupernatant was quickly removed and transferred to another sample tubeand 15 microliter loading dye was added. This sample was loaded on asequence gel together with the earlier mentioned samples.

All above mentioned samples were analysed on a denaturing 6%polyacrylamide gel.

5. Result

FIG. 7 contains:

AFLP reactions prepared with a MseI adapter- and AFLP primers containinga GsuI site:

-   Lane 1: contains a 10 basepair size ladder as a reference for    fragment size; fragment sizes in basepairs are indicated on the    left.-   Lane 2: sample IR20, AFLP reaction E35/00s45thio (GsuI site) in    which the E35 primer was labelled with 33P.-   Lane 3: sample 6383, AFLP reaction E35/00s45thio (GsuI site) in    which the E35 was labelled with 33P.-   Lane 4: sample IR20, AFLP reaction E35/00s45thio (GsuI site) in    which the 00s45thioprimer was labelled with 33P.-   Lane 5: sample 6383, AFLP reaction E35/00s45thio (GsuI site) in    which the 00s45thioprimer was labelled with 33P.-   Lane 6: sample IR20, AFLP reaction E35/00s45thio (GsuI site) in    which the E35 primer was labelled with 33P, followed by digestion    with Exonuclease I and GsuI.-   Lane 7: sample 6383, AFLP reaction E35/00s45thio (GsuI site) in    which the E35 primer was labelled with 33P, followed by digestion    with Exonuclease I and GsuI.-   Lane 8: sample IR20, AFLP reaction E35/00s45thio (GsuI site) in    which the 00s45thioprimer was labelled with 33P, followed by    digestion with Exonuclease I and GsuI.-   Lane 9: sample 6383, AFLP reaction E35/00s45thio (GsuI site) in    which the 00s45thioprimer was labelled with 33P, followed by    digestion with Exonuclease I and GsuI.-   Lane 10: sample IR20, AFLP reaction E35/00s45thio (GsuI site) in    which the E35 primer was labelled with 33P, followed by digestion    with Exonuclease I, GsuI and T7 Gene 6 Exonuclease.-   Lane 11: sample 6383, AFLP reaction E35/00s45thio (GsuI site) in    which the E35 primer was labelled with 33P, followed by digestion    with Exonuclease I, GsuI and T7 Gene 6 Exonuclease.-   Lane 12: sample IR20, AFLP reaction E35/00s45thio (GsuI site) in    which the 00s45thioprimer was labelled with 33P, followed by    digestion with Exonuclease I, GsuI and T7 Gene 6 Exonuclease.-   Lane 13: sample 6383, AFLP reaction E35/00s45thio (GsuI site) in    which the 00s45thio primer was labelled with 33P, followed by    digestion with Exonuclease I, GsuI and T7 Gene 6 Exonuclease.-   Lane 14: sample IR20, AFLP reaction E35/00s45thio (GsuI site) in    which the E35 primer was labelled with 33P, followed by digestion    with Exonuclease I, GsuI and T7 Gene 6 Exonuclease and then followed    by purification using a nucleotide removal column.-   Lane 15: sample 6383, AFLP reaction E35/00s45thio (GsuI site) in    which the E35 primer was labelled with 33P, followed by digestion    with Exonuclease I, GsuI and T7 Gene 6 Exonuclease and then followed    by purification using a nucleotide removal column.-   Lane 16: sample IR20, AFLP reaction E35/00s45thio (GsuI site) in    which the 00s45thioprimer was labelled with 33P, followed by    digestion with Exonuclease I, GsuI and T7 Gene 6 Exonuclease and    then followed by purification using a nucleotide removal column.-   Lane 17: sample 6383, AFLP reaction E35/00s45thio (GsuI site) in    which the 00s45thioprimer was labelled with 33P, followed by    digestion with Exonuclease I, GsuI and T7 Gene 6 Exonuclease and    then followed by purification using a nucleotide removal column.    FIG. 8 contains:    AFLP reactions prepared with a MseI adapter- and AFLP primers    containing a BsgI site:-   Lane 1: contains a 10 basepair ladder as a reference for fragment    size; fragment sizes in basepairs are indicated on the left.-   Lane 2: sample IR20, AFLP reaction E35/00s24bio (BsgI site) in which    the E35 primer was labelled with 33P.-   Lane 3: sample 6383, AFLP reaction E35/00s24bio (BsgI site) in which    the E35 primer was labelled with 33P.-   Lane 4: sample IR20, AFLP reaction E35/00s24bio (BsgI site) in which    the 00s24bio primer was labelled with 33P.-   Lane 5: sample 6383, AFLP reaction E35/00s24bio (BsgI site) in which    the 00s24bio primer was labelled with 33P.-   Lane 6: sample IR20, AFLP reaction E35/00s24bio (BsgI site) in which    the E35 primer was labelled with 33P, after digestion with    Exonuclease I and BsgI.-   Lane 7: sample 6383, AFLP reaction E35/00s24bio (BsgI site) in which    the E35 primer was labelled with 33P, after digestion with    Exonuclease I and BsgI.-   Lane 8: sample IR20, AFLP reaction E35/00s24bio (BsgI site) in which    the 00s24bio primer was labelled with 33P, after digestion with    Exonuclease I and BsgI.-   Lane 9: sample 6383, AFLP reaction E35/00s24bio (BsgI site) in which    the 00s24bio primer was labelled with 33P, after digestion with    Exonuclease I and BsgI.-   Lane 10: sample IR20, AFLP reaction E35/00s24bio (BsgI site) in    which the E35 primer was labelled with 33P, after digestion with    Exonuclease I and BsgI, followed by isolation of the oligonucleotide    tags using magnetic streptavidin beads.-   Lane 11: sample 6383, AFLP reaction E35/00s24bio (BsgI site) in    which the E35 primer was labelled with 33P, after digestion with    Exonuclease I and BcgI digested DNA I, followed by isolation of the    oligonucleotide tags using magnetic streptavidin beads.-   Lane 12: sample IR20, AFLP reaction E35/00s24bio (BcgI digested DNA    I site) in which the 00s24bio primer was labelled with 33P, after    digestion with Exonuclease I and BsgI, followed by isolation of the    oligonucleotide tags using magnetic streptavidin beads.-   Lane 13: sample 6383, AFLP reaction E35/00s24bio (BsgI site) in    which the 00s24bioprimer was labelled with 33P, after digestion with    Exonuclease I and BsgI, followed by isolation of the oligonucleotide    tags using magnetic streptavidin beads.    FIG. 7 shows:-   1) Labelling of either E35 or 00s24thio results in a comparable    fingerprint as expected in lanes 2 and 4, and lanes 3 and 5 with    some mobility changes due too sequence differences between the 33P    labelled strands of the fragments.-   2) Fragment sizes in lanes 6 and 7 are reduced by 29 nucleotides    after digestion with GsuI compared to the fragment sizes in lanes 2    and 3.-   3) As expected almost all fragments are digested by GsuI resulting    in a strong fragment of 29 nucleotides in lanes 8 and 9.-   4) Lanes 10 and 11 show that the by GsuI digested fragments seen in    lanes 6 and 7 are as expected digested by T7 Gene 6 Exonuclease.-   5) The 33P labelled 00s24thio containing fragments are as expected    unaffected by the digestion of T7 Gene 6 Exonuclease as seen in    lanes 12 and 13.-   6) After purification with the nucleotide removal kit the fragments    of 29 nucleotides are still present as seen in lanes 16 and 17.    FIG. 8 shows:-   1) Labelling of either E35 or 00s24bio results in a comparable    fingerprint as expected in lanes 2 and 4, and lanes 3 and 5 with    some mobility changes due too sequential differences between the 33P    labelled strands of the fragments and the presence of a biotin    molecule (which results in a lower mobility of the fragments).-   2) Fragment sizes in lanes 6 and 7 are reduced with 29 nucleotides    after digestion with BsgI compared when with the fragment sizes in    lanes 2 and 3.-   3) As expected almost all fragments are digested by BsgI resulting    in a strong fragment of 29 nucleotides (which due to the presence of    a biotin molecule migrates as a fragment with a mobility of 34    nucleotides) in lanes 8 and 9.-   4) After purification by the streptavidin coated magnetic beads the    fragments of 29 nucleotides are still present in lanes 12 and 13.

EXAMPLE V

Generation of Oligotags by AFLP Reactions Using a Type IIS RestrictionEnzyme, Adapter and Amplification Primer.

1. Biological Material

The biological material used are the two parental lines (samples IR20and 6383) from a F2 population of Rice. The parental lines were acquiredfrom the IRRI in the Philippines.

2. BcgI AFLP Template Preparation, Preamplification and SelectiveAmplification.

2.1 Template Preparation Using BcgI.

Template preparations were conducted according to the standard protocols(Vos et al., Nucleic Acids Research 23: no 21, pp. 4407–4414, and patentapplication EP0534858) with the exception that the BcgI restrictionenzyme was inactivated by incubating the restriction digestion mix for10 minutes at 70° C. prior to the ligation of the adapters. Thesequences of the adapters, preamplification primers and selectiveamplification primers are as follows:

BcgI adapter sequence1:

00s25: 5′-ctcgtagactgcgtaccNN-3′ (SEQ ID NO.32) 00s26:5′-ggtacgcagtctacgag-3′ (SEQ ID NO.33)

With N being any of the four deoxynucleosides A, C, G or T

BcgI adapter sequence2:

00s27: 5′-gacgatgagtcctgagaNN-3′ (SEQ ID NO.34) 00s28:5′-tctcaggactcatcgtc-3′ (SEQ ID NO.35)

With N being any of the four deoxynucleosides A, C, G or T

BcgI + 1 preamplification primer on adapter sequence 1: 00s29:5′-ctcgtagactgcgtacca-3′ (SEQ ID NO.36) BcgI + 2 preamplification primeron adapter sequence 2: 00s35: 5′-gacgatgagtcctgagacc-3′ (SEQ ID NO.37)BcgI + 3 amplification primer on adapter sequence 1: 00s31bio:5′-bio-ctcgtagactgcgtaccaca-3′ (SEQ ID NO.38)(bio refers to a biotin molecule coupled to the 5′-end of the primer)

BcgI +4 amplification primers on adapter sequence 2:

00s41: 5′-gacgatgagtcctgagaccac-3′ (SEQ ID NO.39) 00s42:5′-gacgatgagtcctgagaccag-3′ (SEQ ID NO.40)2.2 Preamplifications.

Preamplification reactions were set up according to the standardprotocols with the exception that the above mentioned primers (00s29 and00s35) were used and that the thermal cycling protocol was adapted foramplification of small fragments.

The thermal cycling protocol used was 40 cycles with 1 sec 94° C. and 5sec 56° C.

2.3 AFLP Selective Amplification.

AFLP reactions were radioactively labelled with 33P and resolved on astandard AFLP (sequence) gel.

The set-up of the selective amplification reactions was according thestandard protocol with the exception that the above mentioned primerswere used in the following combinations:

Combination 1: 00s31bio/00s41

Combination 2: 00s31bio/00s42

The selective amplification thermal cycling protocol was adapted foramplification of small fragments.

The selective thermal cycling protocol used was 13cycles (1 sec 94° C.and 5 sec 65° C.) in which the 65° C. is lowered to 56° C. followed by23 cycles (1 sec 94° C. an sec 56° C.).

3. Preparing AFLP Reactions with Double Stranded Fragments.

After the selective amplification reaction in a 20 microliter volumewith primer sequences described in section 2.3, an identical amount of20 microliter selective amplification reagents was added to yield a 40microliter total volume, and an extra cycle of PCR amplification wasconducted according to the thermal cycle profile 1 sec 94° C. and 5 sec56° C., to convert all AFLP fragments to double stranded DNA fragments.

4. Preparation of AFLP Oligonucleotide Tags.

Following the preparation of double stranded AFLP reactions as describedin sections 2 and 3 above, a 5 microliter sample was taken from eachtube and mixed with 5 microliter loading dye as a control prior to AFLPoligonucleotide tag preparation.

In a tube 5 microliter double stranded AFLP reaction product as mixedwith 15 microliter, contains 5 microliter magnetic streptavidin coatedbeads in STEX, and incubated for 30 minutes at room temperature withgentle agitation. After this incubation the beads were concentrated withthe magnetic particle concentrator and washed sequentially twice with100 microliter STEX and once with 100 microliter ddH2O and reconstitutedin 10 microliter ddH2O. The oligonucleotide tags are incubated for 5minutes at 95° C. and after concentration on a magnetic particleconcentrator the supernatant was quickly removed and transferred toanother sample tube and 10 microliter loading dye was added. This samplewas loaded on a sequence gel along side the earlier mentioned samples.

All above-mentioned samples were analysed on a denaturing 6%polyacrylamide sequence gel.

5. Results

FIG. 9 contains:

AFLP reactions prepared from templates prepared with the BcgIrestriction enzyme:

-   Lane 1, 6, 7 and 12: contain a 10 basepair size ladder as a    reference for fragment size; fragment sizes in basepairs are    indicated on the right.-   Lane 2: sample IR20, AFLP reaction 00s40/00s31bio in which the 00s40    primer was labelled with 33P.-   Lane 3: sample 6383, AFLP reaction 00s40/00s31bio in which the 00s40    primer was labelled with 33P.-   Lane 4: sample IR20, AFLP reaction 00s40/00s31bio in which the 00s40    primer was labelled with 33P followed by isolation of the    oligonucleotide tags using magnetic streptavidin beads.-   Lane 5: sample 6383, AFLP reaction 00s40/00s31bio in which the 00s40    primer was labelled with 33P followed by isolation of the    oligonucleotide tags using magnetic streptavidin beads.-   Lane 8: sample IR20, AFLP reaction 00s41/00s31bio in which the 00s41    primer was labelled with 33P.-   Lane 9: sample 6383, AFLP reaction 00s41/00s31bio in which the 00s41    primer was labelled with 33P.-   Lane 10: sample IR20, AFLP reaction 00s41/00s31bio in which the    00s41 primer was labelled with 33P followed by isolation of the    oligonucleotide tags using magnetic streptavidin beads.-   Lane 11: sample 6383, AFLP reaction 00s41/00s31bio in which the    00s41 primer was labelled with 33P followed by isolation of the    oligonucleotide tags using magnetic streptavidin beads.    1. FIG. 9 shows:-   1) The adapted amplification profile shows clear amplification of    the 70 nucleotide fragments as shown in lanes 2, 3, 8 and 9.-   2) After purification with streptavidin coated magnetic beads,    fragments of 70 nucleotides are still present in lanes 4, 5, 10 and    11.

EXAMPLE VI Representation of Alternative Method for GeneratingOligonucleotide Tags with Fixed Length for e.g. Mass-SpectroscopicAnalysis.

Starting with BcgI digested DNA fragments:

5′ nnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnnnn-3′ (SEQ ID NO. 81)3′ nnnnnnnnnnnnGCTnnnnnnACGnnnnnnnnnn-5′ (SEQ ID NO. 82)Adapters X (xxxxxNN) and Y (yyyyNN), each with another core sequence areligated onto the BcgI digested DNA fragments.

xxxxxNNnnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnnnnYYYYY (SEQ ID NO. 83)XXXXXnnnnnnnnnnnnGCTnnnnnnACGnnnnnnnnnnNNyyyyy (SEQ ID NO. 84)The complexity of the DNA mixture is reduced through amplification withselective nucleotides. In the last amplification a primer is used with a5′-biotin molecule.

Bio-xxxxATGnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnTCGYYYYY (SEQ ID NO. 85)XXXXXTACnnnnnnnnnGCTnnnnnnACGnnnnnnnnnAGCyyyyy (SEQ ID NO. 86)Biotinylated fragments are purified from the amplification reactionusing streptavidin coated magnetic beads.

Bio-xxxxxATGnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnTCGYYYYY (SEQ ID NO. 87)XXXXXTACnnnnnnnnnGCTnnnnnnACGnnnnnnnnnAGCyyyyy (SEQ ID NO. 88)The fragments coupled on the magnetic beads are then digested. Only theshort fragments coupled on the beads are digested as these are the onlyfragments which carry a recognition site for the restriction enzyme. Inthis way fragments of fixed length are released from the beads which arein the range for analysis on e.g. MALDI-TOF mass-spectroscopic analysis.

Bio-xxxxxAT   GnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnTCG    YYYYY (SEQ ID NO.89)   XXXXX     TACnnnnnnnnnGCTnnnnnnACGnnnnnnnnnA   GCyyyyy (SEQ ID NO.90)The released short fragments are purified with an nucleotide removalkit.

  GnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnTCG    YYYYY (SEQ ID NO. 91)TACnnnnnnnnnGCTnnnnnnACGnnnnnnnnnA  GCyyyyy (SEQ ID NO. 92)The purified short fragments are analysed. Due to the presence of bothstrands of the BcgI fragments there is a each fragment will result in 2peaks in a Mass spectroscopic analysis. If the presence of both strandsis a problem, during the last amplification step a primer can be usedwhich carries a biotin molecule on it's 5′ prime end and carriesnuclease resistant inter nucleotide bonds like phosphorothioate bonds.Such a primer would look like:5′-biotin-xxxxxATGThe nucleotides A, T and G are separated by nuclease resistant bonds.When this primer is used the with streptavidin coated magnetic beadspurified fragments can be cut with BcgI. One side of the fragment (whichis coupled to the magnetic beads) will only be cut in one of the strandof the fragment whereas the other side of the fragment is cut in bothstrands. This results in the next fragments.

Bio-xxxxxATGnnnnnnnnnCGAnnnnnnTGCnnnnnnnnnTCG    YYYYY (SEQ ID NO. 93)  XXXXXTACnnnnnnnnnnnGCTnnnnnnACGnnnnnnnnnA   GCyyyyy (SEQ ID NO. 94)The underlined nucleotides are separated but remain coupled to the otherstrand at this point. The beads are washed which removes the smallfragments indicated on the right. When the resulting fragments on thebeads are denatured e.g. by alkali or heat treatment, only two types offragments are released. Shown in the same way as above these fragmentslook like below.XXXXX TACnnnnnnnnnGCTnnnnnnACGnnnnnnnnnA (SEQ ID NO. 95)The fragments generated in this way are in the range for optimaldetection by mass-spectroscopic analysis.The skilled person will appreciate that the method of Example VI mayequally be carried out using any of the other “double cutter”restriction endonucleases, i.e. restriction endonucleases which cut attwo sites, different from the recognition site of the restrictionendonuclease. Likewise, other affinity labels than the biotin label maybe applied.

EXAMPLE VII MaldiTOF-Mass Spectrophotometric Analysis of OligonucleotideTags from AFLP Reactions Generated with a MseI AFLP Adaptor and anAmplification Primer Containing a Type IIS Restriction Enzyme Site.

1. Preparation of AFLP Oligonucleotide Tags.

The oligonucleotide tags analysed were generated from the parental line6383 used in example IV with the procedure stated in the example IV withthe exception that a biotinylated primer containing a GsuI restrictionsite was used.

The oligonucleotide tags generated, were analysed by MaldiTOF-Massspectrophotometry of which the result is shown in FIG. 10.

2. Results

FIG. 10 contains a mass spectrum from a MaldiTOF-Mass spectrophotometricanalysis performed on the above generated oligonucleotide tags.

FIG. 10 shows:

1) Clear distinct peaks around the mass of 8615, which is the averagemass of the oligonucleotide tags generated.

2) A total of 20–25 peaks are visible which represent 20–25oligonucleotide tags with different masses.

1. Method for detecting a restriction fragment, comprising the steps of:(A) digesting a starting DNA, with one or more specific restrictionendonucleases, to fragment said starting DNA into a series ofrestriction fragments; (B) ligating the restriction fragments thusobtained with at least one double-stranded oligonucleotide adapter, oneend of which can ligate with one or both of the ends of the restrictionfragments, to thereby produce tagged restriction fragments of thestarting DNA; (C) contacting said tagged restriction fragments underhybridising conditions with at least one oligonucleotide primer; (D)amplifying said tagged restriction fragments hybridised with saidprimers by PCR so as to cause further elongation of the hybridisedprimers along the restriction fragments of the starting DNA to whichsaid primers hybridised; and (E) identifying or recovering the amplifiedor elongated DNA fragment thus obtained; in which the detection of theamplified restriction fragments of step E) is carried out by massspectroscopy, chromatography, or a combination thereof.
 2. Method fordetecting a restriction fragment, comprising the steps of: (A) digestinga starting DNA, with one or more specific restriction endonucleases, tofragment said starting DNA into a series of restriction fragments; (B)ligating the restriction fragments thus obtained with onedouble-stranded oligonucleotide adapter to thereby produce taggedrestriction fragments of the starting DNA; (C) contacting said taggedrestriction fragments under hybridising conditions with at least oneoligonucleotide primer; (D) amplifying said tagged restriction fragmentshybridised with said primers by PCR so as to cause further elongation ofthe hybridised primers along the restriction fragments of the startingDNA to which said primers hybridised; and (E) identifying or recoveringthe amplified or elongated DNA fragment thus obtained; in which: theadapter used in step B) comprises within its sequence a recognition sitefor a IIS restriction endonuclease; and in which: step E) comprises thesteps of: d) restricting the ligated dsDNA with the restrictionendonuclease of the IIS type which recognizes the recognition sitewithin the adapter so as to obtain at least a first and a secondIIS-restricted dsDNA; e1) generating at least one ssDNA from at leastone of the amplified restriction fragments obtained in step D); e2)detecting the at least one ssDNA generated in step e1).
 3. Methodaccording to claim 2, in which the at least one ss DNA generated in stepe1) is detected in step e2) by mass spectroscopy, chromatography, or acombination thereof.
 4. Method according to claim 2, in which at leastone of the primers used in step (C) is resistant to an exonuclease. 5.Method according to claim 2, in which in step e1), the mixture ofamplified restriction fragments obtained in step D) is treated with anexonuclease, optionally after heat denaturation.
 6. Method forgenerating, and optionally detecting, an oligonucleotide that isspecific for a starting nucleic acid, comprising the steps of: a)providing a dsDNA; b) restricting the dsDNA with at least onerestriction endonuclease that restricts the dsDNA at two sites differentfrom the recognition site of said restriction endonuclease, so as toprovide a mixture of restricted fragments, said mixture comprising oneor more fragments that contain the recognition site of the restrictionendonuclease and one or more fragments that do not contain therecognition site of the restriction endonuclease used; c) ligating thefirst dsDNA to a second dsDNA, so as to provide a ligated dsDNA; d)amplifying the mixture of fragments obtained in step c); e) detecting atleast one of the amplified fragments obtained in step d).
 7. Methodaccording to claim 6, in which, in step (e), one or more of theamplified fragments that contain the recognition site of the restrictionendonuclease are detected, optionally after isolation of these fragmentsfrom the amplified mixture obtained in step d).
 8. Method according toclaim 6, in which detection step (e) is carried out on ligated dsDNAobtained in step (c), optionally after specific isolation of one or morethe ligated dsDNA fragments.
 9. Method according to claim 6, in which inthe amplification step d), a biotinylated primer is used.
 10. Methodaccording to claim 6, in which in the amplification step d), anexonuclease resistant primer is used.
 11. Method according to claim 10,in which a ssDNA is generated by treating a strands of the type IISrestricted fragments obtained in step d) with an exonuclease.
 12. Methodaccording to claim 6, in which the amplification of step c) is carriedout using primers that can hybridise with at least part of thenucleotide sequence of the adapters ligated to the ends of therestriction fragments to be amplified and which optionally comprise oneor more selective bases at the 3′-end.
 13. Method according to claim 6,in which the detection of step d) is carried out by mass spectroscopy orchromatography or a combination thereof.
 14. Method for generating, andoptionally detecting, an oligonucleotide that is specific for a startingnucleic acid, comprising the steps of: a) providing a first dsDNA; b)restricting the first dsDNA with at least one restriction endonucleasethat restricts the first dsDNA at two sites different from therecognition site of the restriction endonuclease, so as to provide amixture of restricted fragments, the mixture comprising one or morefragments that contain the recognition site of the restrictionendonuclease and one or more fragments that do not contain therecognition site of the restriction endonuclease; c) ligating therestricted fragments of the first dsDNA as obtained in (b) to a seconddsDNA, in which the second dsDNA contains a recognition sequence for atype IIS restriction endonuclease in close proximity to the end ligatingto the restricted fragment of the first dsDNA, so as to provide aligated dsDNA; d) amplifying the ligated dsDNA obtained in (c); e)digestion of the amplified fragments obtained in (d) with the type IISrestriction endonuclease; f) detecting a fragment obtained in (e). 15.Method for generating, and optionally detecting, an oligonucleotide thatis specific for a starting nucleic acid, comprising the steps of: a)providing a first dsDNA; b) restricting the first dsDNA with at leastone restriction endonuclease that restricts the first dsDNA at two sitesdifferent from the recognition site of the restriction endonuclease, soas to provide a mixture of restricted fragments, the mixture comprisingone or more fragments that contain the recognition site of therestriction endonuclease and one or more fragments that do not containthe recognition site of the restriction endonuclease; c) ligating ds DNAadapter sequences to both ends of the restriction fragments, so as toprovide a ligated dsDNA; d) amplifying the ligated dsDNA obtained in(c); e) digestion of the amplified fragments obtained in (d) with therestriction endonuclease; f) detecting a fragment obtained in (e). 16.Method according to claim 15, in which in step (f) one or more of theamplified fragments that contain the recognition site of the restrictionendonuclease are detected, optionally after specific isolation of atleast one of the amplified fragments.